CyAn ADP with Summit Software User Guide

CyAn ADP with Summit Software User Guide Document Number 0000050 Revision F July 2006

Copyright 2001-2006 Dako. All rights reserved. This document may not be copied in whole or in part or reproduced in any other media without the express written permission of Dako. Please note that under copyright law, copying includes translation into another language. 2 CyAn ADP with Summit Software User Guide

The CyAn ADP Analyzer is the next step in Advanced Digital Processing (ADP) with enhanced detection for flow cytometry. The CyAn ADP High-Performance Flow Cytometer is a research tool engineered for precision analysis of cells, bacteria, and other similarly sized particles. With CyAn ADP, Dako sets a new industry standard with a combination of features never before available on a bench-top analyzer. CyAn ADP gives users three excitation lines with independent, alignmentfree focusing optics, simultaneous nine color and two scatter parameters, analysis rates of 70,000 events per second, a full 9 9 interlaser compensation matrix, and high sensitivity. The result is stable, user-friendly, and flexible technology. The instrument is optimized for cell cycle, kinetics, fluorescent protein work, and multi-color immunophenotyping. Rare-event analysis, such as MHC Dextramer studies, and no-lyse whole blood applications are easily performed on the CyAn ADP. The instrument also provides simplified compensation before, during, and after acquisition with unequaled sensitivity in all fluorescent parameters. CyAn ADP with Summit Software User Guide 3

Contacting Dako Dako should be contacted immediately for assistance in the event of any instrument malfunction. For further information please contact your local Dako office. Australia Tel. 2 9316 4633 Fax 2 9316 4773 Austria Tel. 0800 0800 7153 Fax 0800 0800 7154 Belgium Tel. 016 38 72 20 Fax 016 38 72 21 Canada Tel. 905 858 8510 Fax 905 858 8801 Czech Republic Tel. 420 541 423 710 Fax 420 541 423 711 Denmark Head Office Tel. 44 85 95 00 Fax 44 85 95 95 Sales Tel. 44 85 97 56 Fax 44 85 84 29 France Tel. 1 30 50 00 50 Fax 1 30 50 00 11 Germany Tel. 040 69 69 470 Fax 040 69 52 741 Italy Tel. 02 58 078 1 Fax 02 58 078 292 Japan Tel. 075 211 3655 Fax 075 211 1755 The Netherlands Tel. 020 42 11 100 Fax 020 42 11 101 Norway Tel. 23 14 05 40 Fax 23 14 05 42 Poland Tel. 058-661 1879 Fax 058-661 3390 Spain Tel. 93 499 05 06 Fax 93 499 02 08 Sweden Tel. 08 556 20 600 Fax 08 556 20 619 Switzerland Tel. 041 760 11 66 Fax 041 760 11 77 United Kingdom Tel. (0)1 353 66 99 11 Fax (0)1 353 66 89 89 Technical Support Tel. (0) 1 353 66 99 65 United States of America Carpinteria, California Tel. 805 566 6655 Fax 805 566 6688 Technical Support Tel. 800 424 0021 Customer Service Tel. 800 235 5763 United States of America Fort Collins, Colorado Flow Instrumentation Tel. 800 822 9902 Fax 970 226 0107 4 CyAn ADP with Summit Software User Guide

User Resources For the latest information on Dako products and services, please visit the Dako Web site at http://www.dako.com, and the Dako Flow Cytometry Discussion Group at http://www.dakousa.com/index/support/discussion_groups.htm. Scope This guide provides a detailed discussion of the architecture and operating procedures for the CyAn ADP High-Performance Flow Cytometer. Detailed operating instructions for Summit software can be found in the Summit software online help system. The information contained in this document can be applied to all CyAn ADP products. Disclaimers This document is not a substitute for the detailed operator training provided by Dako or for other advanced instruction in general cytometric techniques. It is essential that the operator have a working knowledge of Microsoft Windows XP or current Operating System prior to using this guide. Dako also recommends the use of all of the networking services provided in the individual operator s laboratory. Although daily maintenance and routine instrument adjustments are discussed here, your local Dako Technical Service Group should be contacted immediately for assistance in the event of any instrument malfunction. Trademarks Microsoft and Windows XP are registered trademarks of Microsoft Corporation. Macintosh is a registered trademark of Apple, Inc. MoFlo is a registered trademark of Dako. CyAn, Summit, SpectrAlign, and SpectraComp are trademarks of Dako. All other trade names and trademarks are the property of their respective holders. CyAn ADP with Summit Software User Guide 5

Table of Contents Section 1 Safety... 9 General Hazard Avoidance... 10 Biohazard Avoidance... 11 Laser Safety... 12 Section 2 Installation... 15 CyAn ADP Installation Requirements... 15 General Laboratory Information... 15 CyAn ADP Power Configuration and Consumption... 17 CyAn ADP Instrument Configurations... 17 Section 3 System Overview... 19 CyAn ADP Features... 19 CyAn ADP Subsystems... 20 Fluidics... 21 Optics... 22 Electronics... 25 Peripheral Devices... 26 Software...26 Summit Software Features... 27 Section 4 Startup and Shutdown Procedures... 29 Required Reagents for Startup and Shutdown... 29 CyAn ADP Startup... 29 Daily Decontamination Procedure... 32 Shutdown Procedure... 33 Section 5 Sheath Management System... 35 Sheath Management System Indicators... 35 Changing Out Sheath and Waste Containers... 41 Replacing Cleaner Fluid... 42 Section 6 Summit Software Basics... 43 Acquire and Analyze up to 10 Million Events... 43 Create a New Database... 44 Open an Existing Database... 46 Create a Histogram... 47 Create a Dot Plot... 48 Open FCS (Listmode) Files... 49 Set a Gate from a Single Region... 51 Set a Serial Gate... 52 Define Gate Logic... 55 Shortcut Keys... 57 Section 7 CyAn ADP Maintenance... 59 Daily Preventive Maintenance... 59 Weekly Preventive Maintenance... 59 Reusable Sheath Containers... 59 Head Unit and SMS Cart... 60 Fluidic Lines... 60 Monthly Preventive Maintenance... 60 System Decontamination... 60 Laser Interlock Maintenance Procedure... 61 Biannual Maintenance Procedure... 61 Section 8 Troubleshooting... 63 Section 9 Six-Color Compensation Experiment... 65 Background... 65 Setting up for sample acquisition... 66 Setting Up Histograms for Compensation... 68 6 CyAn ADP with Summit Software User Guide

Manual Compensation... 71 Setting Spillover Percentage for Compensation... 72 Running the Second Sample... 73 Running the Remaining Samples... 75 Running the All Stains Sample... 83 Putting Compensation into Practice... 84 CD3+ T-Lymphocytes... 84 CD 19+ B-Lymphocytes... 84 CD4+/CD3+ T-Lymphocytes (T-Helper cells)... 85 CD8+/CD3+ T-Lymphocytes (T-Cytotoxic/Suppressor cells)... 85 Natural Killer Lymphocytes... 86 Section 10 Auto Compensation... 87 Auto Compensation for Single Positive Controls... 87 Section 11 VisiComp... 91 Using the VisiComp Feature after Compensation... 91 Section 12 WorkList Builder... 93 Overview... 93 Accessing the WorkList Builder... 94 Creating a New Panel... 95 Setting up Tests and Defining Reagents for a Panel... 97 Creating Reagents by Defining Antibody/Fluorochrome Combinations... 98 Deleting Reagents from the WorkList Builder Database... 100 Setting up Tests (Tubes or Samples) within a Panel... 101 Setting up a Panel... 105 Creating Worklists from Defined Panels and Tubes... 107 Setting up Worklists for Multiple Experiments or Specimens... 108 Customizing the WorkList Builder Viewing Area... 110 Edit Columns... 110 Importing a Specimen List from a Text File... 111 Saving and Reusing Worklists... 112 Associating Protocols, Gate/Event Limits, or Other Information to Worklist Tests... 113 Appendix A Approved Cleaners and Disinfectants... 115 Cleaners... 115 Disinfectants for Use in Sample Line*... 115 Disinfectants for Use in Sheath Line**... 115 Disinfectants for Use in the Waste Tank... 115 Appendix B CyAn ADP Consumables and Parts... 117 Appendix C Technical & Instrument Specifications... 119 Appendix D Symbol Definitions... 123 Appendix E Flow Cytometry References... 125 CyAn ADP with Summit Software User Guide 7

8 CyAn ADP with Summit Software User Guide

Section 1 Safety Electrical Safety The Dako instrument product line conforms to international regulations encompassing the accessibility of high voltages by the user. In the United States, each flow cytometer is manufactured to comply with applicable regulations from the American National Standards Institute (ANSI), the Occupational Safety and Health Administration (OSHA), and various state regulatory organizations. Internationally, each CyAn ADP High-Performance Flow Cytometer is manufactured to comply with the International Electrotechnical Commission s Standard for Safety Requirements for Electrical Equipment for Measurement, Control and Laboratory Use (IEC 61010-1) and subsequent amendments. Biohazard Safety The CyAn ADP instrument may be used for the analysis of pathogenic or other harmful agents. These operations can constitute a biohazard for the operator as well as bystanders in the surrounding laboratory space. Dako does not certify any instrumentation for use with any hazardous organism or agent. Dako strongly recommends explicit guidance be obtained from the institutional or company biosafety officer prior to operating this instrument with any potentially hazardous organism or agent. See the Biohazard Avoidance section for more information. WARNING If any biohazardous organism or agent is used in this instrument, the user must inform Dako in writing prior to any field service visit or the return of any part to Dako, or its vendors, for service. Safety of the CyAn ADP user and of all Dako employees is of primary concern. Proper decontamination procedures must be followed for returned parts. WARNING Wear Personal Protective Equipment (PPE) in accordance with your laboratory safety procedures when operating or maintaining this instrument. It is recommended that gloves, a laboratory coat, and safety glasses are worn at all times when using the CyAn ADP. Use of this instrument in a manner other than that specified in this manual may cause impairment of equipment, compromise safety, and/or result in injury. CyAn ADP with Summit Software User Guide 9

General Hazard Avoidance Safety of the CyAn ADP operator is of primary concern. It is important that all operators familiarize themselves with this safety section to avoid all potential hazards associated with the use of the CyAn ADP. Any questions or concerns regarding safety of the instrument should be directed to Dako Colorado immediately. 1. PINCHING: The lid of the CyAn ADP is on hinges that should keep the lid in the UP position when open. It is recommended by Dako that the lid NOT be left open for extended periods of time OR be opened and closed repeatedly to avoid excessive wear on the hinges which may, over time, cause the lid to fall unexpectedly, potentially falling on an operator s hands or fingers, causing injury. Please use caution when the lid is open and do not leave unattended. If the lid of the CyAn ADP becomes loose and will not stay up on its own, please contact Customer Care so it can be repaired. 2. TRIPPING: The Sheath Management System (SMS) is attached to the CyAn ADP head unit by an 8 foot (2.4 meters)long umbilical cord. It is suggested that the SMS cart be placed underneath a laboratory bench to keep it out of the way of foot traffic through the laboratory. If this is not possible, it is recommended that the SMS be placed where it will minimize the likelihood of a tripping hazard. It is suggested that the umbilical cord be run behind the laboratory bench, if possible, to keep the cord from dangling and potentially creating a tripping hazard as well. If the cord must be run down in front of the laboratory bench to the SMS, please use caution when operating or walking past the CyAn ADP to avoid tripping on the umbilical cord. It is suggested that hazard tape be placed on these items to make them highly visible to minimize the possible tripping hazard in cases where laboratory set up is not ideal. If a person trips on either the umbilical cord or SMS cart of the CyAn ADP, please contact Customer Care. 3. CHEMICAL: PPE should be worn at all times when using any part of the CyAn ADP to ensure protection from all potential hazards. Use caution when handling disinfectant products used in the waste tank. It is important that the appropriate type and amount of disinfectant is used in the waste tank to ensure effective inactivation of biologics. Please see Appendix A for a list of approved cleaners and disinfectants. DO NOT use any cleaner or disinfectant that is NOT on this list, as this may cause damage to the system and void your warranty. Please contact Customer Care with any questions regarding chemical usage on your CyAn ADP. 4. ERGONOMICS Computer Use: It is suggested by Dako that the safety officer or designated individual evaluate the operator(s) workstation soon after the installation of the CyAn ADP to ensure proper ergonomic guidelines are followed to minimize any discomfort that may arise from the use of CyAn ADP peripheral equipment. If any operator experiences any physical discomfort when using the CyAn ADP, please contact Dako Customer Care. A regular keyboard and mouse are included with the computer. If desired, an ergonomic keyboard or trackball mouse may be changed out (at the institution s expense) on the computer supplied with the CyAn. If problems arise after installation of alternative peripheral equipment, please contact Customer Care immediately. 5. ERGONOMICS - Lifting: The sheath and waste tank containers used on the SMS cart have a maximum capacity of 20L. When full, either container will weigh approximately 19kg (42 pounds). Use caution when lifting full containers, whether it be removing or replacing it on the cart, to avoid back strain or injury. Proper lifting techniques must always be used when handling heavy items to avoid back injury. If an operator is unable to comfortably lift a container, it is recommended to seek assistance with moving the container to the desired position. It is also suggested that the waste tank be emptied when half full so that it will be 10 CyAn ADP with Summit Software User Guide

easier to handle. If any operator experiences any physical discomfort when handling the sheath or waste containers on the SMS cart, please contact Dako Customer Care. 6. SKIN ABRASION: The sample probe is constructed of stainless steel and the end of the probe is slightly squared off. Accidental direct contact with the end of the probe and the skin could cause an abrasion or cut. Use caution when loading and unloading samples on the probe. PPE should be worn at all times when using any part of the CyAn ADP. Samples of any kind should never be used on the CyAn without gloves being worn. If any injury related to the sample probe occurs, contact Dako Customer Care immediately. Biohazard Avoidance Safety of the CyAn ADP operator is of primary concern. It is important that all operators familiarize themselves with this safety section to avoid all potential biohazards associated with the use of the CyAn ADP. Any questions or concerns regarding biohazard safety and the CyAn ADP should be directed to Dako Customer Care immediately. 1. WASTE TANK: The reusable waste tank is located on the right side of the SMS cart and is labeled with a large black and orange biohazard symbol on the front. Emptying waste fluid generated from the CyAn ADP could be a daily occurrence for some operators. It is expected that all operators have undergone proper blood borne pathogens training provided by the institution of use, or similar authority, prior to using the CyAn. It is pertinent that the appropriate type and quantity of disinfectant is placed in the waste tank to ensure effective inactivation of the biologics in use when the tank is full. Please see Appendix A for a list of approved cleaners and disinfectants for use with the CyAn ADP. Please use caution when emptying the waste tank to avoid splashing of the waste fluid. PPE should be worn at all times when using any part of the CyAn ADP, including emptying the waste tank. Please contact Dako Customer Care if there are any questions or concerns about handling waste fluid generated by the CyAn ADP. 2. SAMPLE WASTE: Exposure to sample waste fluid from clogs in the waste line may occur; however, this is rare. If a clog occurs, exposure to biohazards is likely. PPE should be worn at all times when using the CyAn ADP to protect the operator from unexpected hazards. If exposure to biohazardous material occurs due to a fault of the CyAn ADP, Dako Customer Care should be contacted immediately. 3. SAMPLES: Sample handling is a normal part of the use of the CyAn ADP. It is expected that all operators have undergone proper bloodborne pathogens training provided by the institution of use, or similar authority, prior to using the CyAn ADP. PPE should be worn at all times when using any part of the CyAn ADP. Samples of any kind should not be handled without wearing gloves. Caution should be used when handling any biohazardous samples. Any reagents used on the instrument, or in the preparation of samples for use on the CyAn ADP, should be handled by professional users only. 4. SAMPLE O-Ring: There is an o-ring located in the top interior of the sample injection area. The purpose of this o-ring is to create a seal when a test tube is placed on the sample probe, which results in pressurization of the tube. Over time this o-ring may lose its elasticity or become damaged from repeated contact with polypropylene test tubes. Deterioration of this o-ring may cause test tubes to be loose when placed on the sample probe, leading to the potential for test tubes filled with biohazardous samples falling off, causing exposure to the operator or other laboratory personnel. This occurrence is not likely, but if an operator experiences this condition, Dako Customer Care should be contacted immediately so the o- ring can be replaced. CyAn ADP with Summit Software User Guide 11

5. SAMPLE Waste Manifold: Waste fluid is continuously generated when running the CyAn ADP and is a normal function of this instrument. This waste fluid is routed through a manifold located within the SMS cart. If this part malfunctions, a backup of fluid may occur, resulting in waste fluid dripping through a small hole in the top corner of the sample injection area. Because this waste fluid may contain biohazardous material, caution should be used if this fault is identified; however, this occurrence is highly unlikely. PPE should be worn at all times when using any part of the CyAn ADP to ensure protection of the operator from all potential hazards. If an operator identifies such a leak, Customer Care should be contacted immediately. 6. AEROSOLS: The CyAn ADP is a flow cytometry analyzer and has no sorting capabilities. No aerosols are generated under normal operating conditions. In the event of extreme over pressurization of the system, there is a remote chance that sample and/or waste fluid could exit orifices at a rate which could potentially create aerosols; however, this has never been experienced. If at any time an operator identifies a potential overpressurization problem, Customer Care should be contacted immediately. 7. SAMPLE PROBE: The sample probe is constructed of stainless steel and the end of the probe is slightly squared off. Accidental direct contact with the end of the probe and the skin could cause an abrasion or cut. Because biohazardous samples are run through the sample probe, there is a chance that if the skin is broken due to direct contact with the end of the sample probe, an operator could be directly exposed to biohazardous material. Use caution when loading and unloading samples on the probe. PPE should be worn at all times when using any part of the CyAn ADP. Samples of any kind should never be used on the CyAn ADP without gloves being worn. If any injury related to the sample probe occurs, Dako Customer Care should be contacted immediately. Laser Safety WARNING Wear Personal Protective Equipment in accordance with your laboratory safety procedures when operating or maintaining this instrument. Use of this instrument in a manner other than that specified in this manual may cause impairment of equipment, compromise safety, and/or result in injury. CAUTION Use of controls or adjustments or performance of procedures other than those specified in this manual may result in hazardous radiation exposure. This radiation will be in the visible regions of the electromagnetic spectrum. Do not attempt to defeat the interlocks or open covers or panels retained with screws. The CyAn ADP instrument conforms to international regulations encompassing laser safety. The CyAn ADP is a Class 1 laser device. CLASS 1 LASER PRODUCT IEC/EN 60825-1/A2:2001 12 CyAn ADP with Summit Software User Guide

This designation indicates no hazardous laser energy is accessible to the user during normal operation or during a failure mode. Note Because the CyAn ADP has been designed to operate as a Class 1 Laser Device, the instrument must be operated with all light containment tubes in place and all protective light seals intact. Most laser components of the CyAn ADP flow cytometer are Class IIIB to Class IV. As such, these lasers have the potential to cause injury. There is one small viewing window inside the CyAn ADP, but its purpose is to be used only by trained Dako personnel during service or repair. A safety interlock prevents operators from viewing laser light through this viewing window under normal operating conditions. Certification of regulatory compliance of these lasers often requires significant involvement from the institutional or company laser-safety officer. Dako does not provide regulatory certification for the individual lab, but can offer assistance in laser safety matters. All access plates and other user accessible points of potential laser exposure are clearly designated on the CyAn ADP instrument with the labels illustrated in figures 1.1, 1.2, and 1.3 below. Interlocks are designed to prevent accidental irradiation of the operator. The user should not defeat these interlocks. Any questions or concerns regarding laser safety of the CyAn ADP should be directed to Dako Customer Care. CyAn ADP with Summit Software User Guide 13

Figure 1.1: Interior Laser Warning Label Locations 14 CyAn ADP with Summit Software User Guide

Section 2 Installation CyAn ADP Installation Requirements IMPORTANT Your Dako representative is responsible for uncrating, installing, and initial set up of the CyAn ADP. General Laboratory Information Avoid placing the CyAn ADP directly below heating and air conditioning vents or fans in order to prevent the possible adverse effects of temperature fluctuation, vibration, and dust. Table 2.1: CyAn ADP Installation Requirements General Requirements Power Requirements Lab Bench Service Access Phone Installation Category 100V 230V, 600VA(W), 50/60 Hz Stable bench/table top to support the CyAn ADP, one monitor, keyboard, and a mouse pad 45.7 cm (18 in) minimum around instrument components Location near CyAn ADP for contacting technical support II Pollution Degree 2 Internet Access Dimensions (not including Auxiliary Components) Internet Service Provider or LAN connection for downloading software updates Height front cover closed - 39.1 cm (15.4 in) front cover open 72.1 cm (28.4 in) Width - 33.3 cm (13.1 in) Depth - 49.8 cm (19.6 in) with clearance for cables 62.5 cm (24.6 in) Weight - 36.3 kg (80 lbs) CyAn ADP with Summit Software User Guide 15

Auxiliary Components Sheath Management System (with casters): Houses sheath container, waste container, cleaner fluid container, air compressor, and vacuum Summit Workstation Optional Uninterruptible Power Supply Height 61.3 cm (24.2 in) Width - 73.3 cm (28.9 in) Depth 61.9 cm (24.4 in) Weight 22.6 kg (50 lbs) Height 42.9 cm (16.9 in) Width 19.1 cm (7.5 in) Depth 45.7 cm (18.0 in) Weight 10.5 kg (23 lbs) Height 20.3 cm (7.9 in) Width 14.7 cm (5.7 in) Depth 44.5 cm (17.5 in) Weight 20 kg (44 lbs) Operating Environment Ambient Temperature Relative Humidity 15 to 30 C (59 to 86 F) For optimum performance maintain at +/- 2 C 20 to 80% RH (non-condensing) 16 CyAn ADP with Summit Software User Guide

CyAn ADP Power Configuration and Consumption Avoid placing the CyAn ADP directly below heating and air conditioning vents or fans in order to prevent the possible adverse effects of temperature fluctuation, vibration, and dust. CyAn ADP Instrument Configurations Instrument Options Laser Configuration Options Code No. Lasers Parameters Colors 488 nm 635 nm 405 nm CY 200* 2 9 7 5 2 - CY 201* 3 11 9 5 2 2 * 30: 110V / 31: 230V / 33: 100V CyAn ADP with Summit Software User Guide 17

18 CyAn ADP with Summit Software User Guide

Section 3 System Overview The CyAn ADP is a state-of-the-art flow cytometer that utilizes one or more laser excitation sources to analyze biological cells, beads, or other microscopic particles as they are transported through an interrogation point in single file. Information can be gathered from large numbers of particles in a relatively short time so that populations can be studied or differentiated from other populations using simple to complex statistical methods. Physical and biochemical characteristics that can be measured using this instrument include but are not limited to forward scatter (sizerelated), side scatter (morphology-related), fluorescence from tagged (stained) cells/particles, and auto fluorescence from non-stained cells/particles. CyAn ADP Features The CyAn ADP High-Performance Flow Cytometer is a research tool engineered for precision analysis of cells, bacteria, and other similarly sized particles. With CyAn ADP, Dako sets a new industry standard with a combination of features never before available on a bench-top analyzer. CyAn ADP gives users three excitation lines with independent, alignment-free focusing optics, simultaneous nine color and two scatter parameters, analysis rates of 70,000 events per second, a full 9 9 interlaser compensation matrix, and high sensitivity. The result is stable, user-friendly, and flexible technology. The instrument is optimized for cell cycle, kinetics, fluorescent protein work, and multi-color immunophenotyping. Rare-event analysis, such as MHC Dextramer studies, and no-lyse whole blood applications are easily performed on the CyAn ADP. The instrument also provides simplified compensation before, during, and after acquisition with unequaled sensitivity in all fluorescent parameters. The main features of CyAn ADP include: Walk-Up Operation CyAn ADP offers walk-up operation that puts even a novice user at ease. With no optical alignment necessary, the user simply places a 5 ml sample tube in the tube holder and chooses the desired protocol in Summit software. The software is used to control instrument settings. Bench-Top Configuration The CyAn ADP was designed with today s laboratories in mind. The CyAn ADP measures 30 cm wide x 45 cm deep x 40 cm high. Summit Software Summit software, Dako s premier cytometry software product, offers full control of all CyAn ADP functions, coupled with robust user- and application-specific data acquisition, analysis, storage, reduction, and retrieval. Summit software controls all instrument parameters in an intuitive MS Windows -based drag-and-drop environment using simple protocol-based menus. Summit CyAn ADP with Summit Software User Guide 19

software also provides high-content data acquisition, database capabilities, and open architecture for data exchange. Additional features include off-line analysis and easy page layout. Safety The CyAn ADP High-Performance Cell/Bead Analyzer has been designed to incorporate the highest level of laboratory and operator safety. CyAn ADP Subsystems CyAn ADP has four key subsystems that form a powerful research tool. The four subsystems are: Fluidics Optics Electronics Software Figure 3.1: CyAn ADP Functional Block Diagram 20 CyAn ADP with Summit Software User Guide

A functional block diagram for the CyAn ADP High-Performance Flow Cytometer is shown in figure 3.1. The fluidics system pressurizes the system and transports particles to the interrogation point. Lasers are used as excitation sources and their beams, along with the ensuing scatter and fluorescence, are directed within the optical system. Electronics are used to power and control the instrument functions. The software provides the user interface for the above fluidics, optical, and electronic hardware, and the functions to acquire, analyze, and store data associated with the particles. Fluidics The Sheath Management System controls the transfer of sheath fluid, waste, and cleaner fluid throughout the CyAn ADP system. Sheath fluid is housed in either a replaceable 20 liter cubitainer or 20 liter plastic carboy. Waste is contained in a 20 liter plastic carboy. Cleaner fluid is contained in a 5 liter cubitainer. The Sheath Management System provides improved sheath pressure stability and prevents bubbles from entering the system when the sheath container is changed. The built-in cleaner fluid cubitainer allows the user to easily clean and rinse the headunit sheath path using Dako Clean and Rinse solution and sheath or DI water as rinsing fluid. The 20 liter sheath and waste containers provide approximately 24 hours of system run time. Figure 3.2 outlines the key fluidics components of CyAn ADP. In general, flow of air and fluids follows a path left to right in the diagram. An air compressor provides pressure for propulsion of the sample, cleaner, and sheath fluids. Air regulators condition and stabilize the pressure source prior to sheath and cleaner reservoirs and sample vessel. A number of electrically controlled valves control the flow state of the system and provide a means for cleaning and debubbling, in addition to routine flow conditions for sample analysis. Sample is forced through tubing, is introduced to the flow cell, and is fluidically focused by sheath fluid. This hydrodynamic focusing effect causes individual particles to be introduced in single file to each of the sequential laser beams. While a pinch valve is used to allow the flow of sample, the rate of sample flow is controlled by adjusting the over-pressure (differential pressure) of an electrically controlled air regulator relative to sheath pressure. Waste is drawn by a vacuum pump to a holding container for disposal. CyAn ADP with Summit Software User Guide 21

Figure 3.2: CyAn ADP Fluidics Block Diagram Optics Three excitation sources are included on the CyAn ADP optical bench. Each path has its own independent, unique steering and focusing elements to provide optimal excitation of particles at the interrogation point. Each laser beam is focused to the quartz flow cell, where particles are transported past the laser beams. Figure 3.3: CyAn ADP Optics Block Diagram 22 CyAn ADP with Summit Software User Guide

The focused beams are separated vertically to ensure minimal optical crosstalk between detection paths. Further, the signals that are detected as particles that traverse the beams are electronically gated to reduce unwanted signal interference between parameters. A high numerical aperture custom achromatic objective is used to collect scatter and fluorescence and to generate an image of the interrogation point for each of three apertures (spatial filters). The apertures improve signal to noise by preventing unwanted scatter around the excitation region from entering the detector block. Light that transmits through the spatial filters is collimated and spectrally filtered on its way to the appropriate photomultiplier tubes (PMT) for detection. CyAn ADP with Summit Software User Guide 23

Figure 3.4 illustrates the location, position, and orientation of the optical filters and PMTs within the detection block assembly of the CyAn ADP. Dichroic filters are located at 45 degrees to the direction of light propagation, while emission filters are located at 90 degrees to the light path. Filter sticks are interchangeable, thus allowing custom configurations to be implemented. Please contact Dako to order dichroic filter sets. 488 nm Laser Laser 1 FL4 SSC FL1 FL2 FL3 FL5 SSC FITC GFP PE PE- TxRed PI PE- Cy5 PerCP 7AAD PE- Cy7 Photomultiplier Tubes 488/10 530/40 575/25 613/20 680/30 750LP Emission Filters R95% 545DLP 595DLP 640DLP 730DLP mirror Dichroic Filters 650DLP 730DLP mirror 485DLP mirror 665/20 750LP 450/50 530/40 Emission Filters Fluorescence & Scatter from Interrogation Point APC APC- Cy7 CAS B CFP DAPI Pacific Blue CAS Y FL9 FL8 FL6 FL7 * Filter configurations are easily modified to accommodate different applications. 635 nm Laser 405 nm Laser Figure 3.4: CyAn ADP Detector Block and Optical Filter Layout 24 CyAn ADP with Summit Software User Guide

Electronics CyAn ADP has an array of electronic components. The PC/workstation is used for instrument control, status, and data acquisition functions and communicates with the CyAn ADP instrument through a high-speed serial link. Housed within the instrument is a state-of-the-art electronic chassis that provides a communication backplane/bus with a number of available card slots to allow modular connection of key electronic control and sensing components. These components include trigger and signal processing, and multi-function input/output. Peripheral to the electronic chassis, but within the instrument are a number of devices that can be grouped as fluidics control and sense (pumps, regulators, valves, and sensors), laser/shutter control and sense, PMT voltage control, and PMT signal. The multifunction I/O card is used for controlling these peripheral devices and sensing instrument status. The PMT and photodiode detectors convert light emitted from particles excited at the interrogation point, into electrical signals. These signals are input to the trigger and signal processing cards. Amplification, analog to digital conversion, and sampling techniques provide quantitative measurements including peak, area/integral, log, and pulse width for a given triggered particle event. CyAn ADP with Summit Software User Guide 25

Peripheral Devices The CyAn ADP instrument includes the following peripheral devices: Summit Workstation with a printer, and the Sheath Management System (SMS). The SMS houses a sheath container, waste container, cleaner cubitainer, reusable plastic carboy, level indicators, in-line sheath filter, and compressor/vacuum pump. Software CyAn ADP uses Summit software for instrument control, data acquisition, and subsequent data analysis. Summit software is the Windows user interface for the entire Dako flow cytometry product line. Summit software offers users complete control of the CyAn ADP instrument at varying levels of complexity, depending on their needs. Summit software, on CyAn ADP with all parameters enabled, has the capability to acquire, save, and analyze up to 10 million events (see page 43). The instrument control panel (figure 3.5) provides access to laser control, event rate settings, system maintenance functions such as clean and rinse, and Sheath Management System functions. The sample parameters panel (figure 3.6) has software controls for adjusting parameter settings such as threshold, PMT voltage, and gain. User documentation for Summit software is available from the Help menu. Figure 3.6: Sample Parameters Panel Figure 3.5: Instrument Control Panel 26 CyAn ADP with Summit Software User Guide

Summit Software Features Summit software offers compensation, flexibility, intelligence, organization, and intuitiveness. Figure 3.7: Summit Software Desktop Display Compensation Summit software performs full inter-laser compensation of fluorescence parameters. Data saved as a sample file in Summit software will retain all compensation values as well as the uncompensated data for each parameter. Flexibility Summit software offers flexibility throughout the program to allow the data to be gathered and manipulated in ways that best suit the application and user. For example, virtually any logical expression can be defined using any number of regions in AND and NOT combinations for generating statistics, including non-rectangular regions with an almost limitless number of vertices. In addition, any and all parameters available with the CyAn ADP High-Performance Flow Cytometer can be selectively stored, including the ability to disable non-contiguous groups of parameters. Eliminating unnecessary parameters increases the display performance during acquisition and reduces data storage needs. CyAn ADP with Summit Software User Guide 27

Intelligence While performing batch analysis, Summit software's intelligent parameter matching feature dynamically determines the correct parameter to display for a sample based on antibody, stain, name, parameter type, channel, or a combination of these. Organization Summit software s sample viewer tracks samples in user-definable folders. Tests and controls with different parameters can be grouped, and samples can be dragged and dropped between folders to help organize information. Statistics for each gate or region are automatically available under each histogram. 28 CyAn ADP with Summit Software User Guide

Section 4 Startup and Shutdown Procedures Required Reagents for Startup and Shutdown Dako Decontamination Solution, catalog # S2324 Dako Clean and Rinse Solution, catalog # S2323 CyAn ADP Startup Note The Quick Start Guide for the CyAn ADP (Document Number. 0000023) is available for a quick reference for Startup and Shutdown procedures. All CyAn ADP users: 1. Log on to the Summit Workstation. 2. Open Summit software by double-clicking the Summit icon on the Windows desktop. 3. Select an existing database or create a new one, and then click OK. CyAn ADP with Summit Software User Guide 29

4. Click the Instrument tab to view the CyAn Control Panel. 5. Check the status of the sheath container and the waste container by viewing the Sheath and Waste levels in the CyAn Control Panel. Make sure the sheath container is at least half full and the waste container is at least half empty. If necessary, fill the sheath container and empty the waste container as described in Changing Out Sheath and Waste Containers on page 41. WARNING Remove the waste container and the sheath container from the cart before emptying or filling. Dispose of the contents of the waste container in accordance with local, state, and federal regulations. WARNING Do not drop the waste or sheath container on the Sheath Management System. Doing so may result in improper calibration of the load cells. 6. Click Startup on the Cyan Control Panel. 7. On the CyAn Control Panel, make sure the desired lasers are checked On. 30 CyAn ADP with Summit Software User Guide

8. Open the required laser shutters by clicking the Closed buttons. These buttons should now read Opened. 9. Allow a minimum of 30 minutes for the lasers to stabilize. 10. It is recommended to perform a Clean and Rinse cycle while the instrument is warming up. Before running the Clean and Rinse cycle, make sure you have enough sheath fluid and cleaner fluid to complete the cycle. On the CyAn Control Panel, click the Sys Clean button A message box will warn you that the Clean and Rinse process will take 10-15 minutes. Click OK to continue. The clean cycle takes seven minutes to complete and the rinse cycle takes seven minutes to complete. 11. Repeat the clean cycle using a tube of DI water. 12. Open your QC file protocol generated by your laboratory, place a tube with SpectrAlign beads (10 6 concentration) on the sample probe, and acquire (F2) at a low event rate (~100 eps). Verify that the data is within your daily QC specification. Dako recommends the following CV specifications for instrument calibration. Parameter FITC < 3.0 PE < 3.0 PE-Texas Red < 3.5 PE-Cy5 < 4.5 PE-Cy7 < 6.5 Pacific Blue < 6.0 Cyan Yellow < 6.0 APC < 6.0 APC-Cy7 < 6.0 Target CV 13. If QC data is within specification, you are ready to run samples. If not, click Debubble on the CyAn Control Panel. Rerun the SpectrAlign beads. If necessary, repeat Debubble and then recheck SpectrAlign beads. CyAn ADP with Summit Software User Guide 31

IMPORTANT After the lasers have stabilized, do not power them down; leave them powered for the duration of CyAn ADP operations for the day. If the lasers are powered down, you must allow another 30 minute stabilization period before running samples again. To conserve sheath fluid, you can shut down the SMS by clicking the Fluidics off button in the CyAn Control Panel. If you have problems with the CyAn ADP or maintenance questions, please contact your local Dako Technical Service Group. Daily Decontamination Procedure It is recommended that after running samples, a decontamination of the sample line be performed on a daily basis. 1. Install a tube containing 3 ml of a 1:5 dilution of Dako Decontamination Solution (S2324) and run for 2 minutes. 2. Remove the tube of Decontamination Solution, replace with a tube of deionized water, and rinse for 2 minutes. 3. Click the Shutdown button on the CyAn Control Panel and perform the steps as indicated in the software. Note: Dako Decontamination Solution contains sodium hypochlorite, which can stain clothing as well as affect the finish of the sample lever arm. Be careful when handling this fluid in order to avoid damage to these items. 32 CyAn ADP with Summit Software User Guide

Shutdown Procedure Dako recommends that Sample Injection Clean and DI Water Clean are enabled for shutdown. Below are the instructions for how to set these criteria. 1. On the Edit menu, click Preferences. 2. Expand the Instrument list item by clicking the + sign. 3. Click CyAn. 4. In the Command Options list, select Shutdown, check the Sample Injection Clean and DI Water Clean check boxes, and then click Save and Close. 5. Click Shutdown on the CyAn Control Panel. This will automatically shut down the lasers and close the laser shutters. When prompted, place a tube full of Clean and Rinse solution on the sample probe and close the lever. Note: If shutting down the instrument for the day, follow the instructions above, but use Decontamination solution instead of Clean and Rinse solution. 6. When prompted, remove the tube, replace with a tube of DI water, and move the sample lever in. CyAn ADP with Summit Software User Guide 33

7. Check the status of the sheath container and the waste container by viewing the Sheath and Waste levels in the CyAn Control Panel. Make sure the sheath container is at least half full and the waste container is at least half empty. If necessary, fill the sheath container and empty the waste container as described in Changing Out Sheath and Waste Containers on page 41. 8. Fill the test tube with DI water, place it on the sample probe and leave the lever out. 9. Close Summit software. Note: Dako recommends waiting 30 seconds after closing Summit software before logging off of the Summit Workstation in order to allow all data sources to close. 10. Log off of the Summit Workstation and turn off the computer. 11. Turn off the computer monitor. 34 CyAn ADP with Summit Software User Guide

Section 5 Sheath Management System Sheath Management System Indicators The Sheath Management System provides 20-24 hours of sample run time. As the level of sheath fluid decreases and the level of waste fluid increases, both Summit software and Light Emitting Diodes (LED) indicators on the Sheath Management System front panel will provide fluid level status information and alerts to the user. SMS indicators can be viewed in the Sheath Management System section of the Instrument tab in the Control Panel. There are indicator lights in the SMS area. When all of the indicator lights are green, all components of the SMS are functioning properly. Figure 5.1 SMS section of Control Panel If an error condition occurs or status changes for a subsystem, an indicator light will change from green to amber or red. When you place your cursor over the indicator light, a message appears that provides a description of the warning and instructions for how to fix the problem. CyAn ADP with Summit Software User Guide 35

The following figures illustrate some of the possible error conditions in the SMS. Figure 5.2 Low sheath fluid warning Figure 5.3 Empty sheath fluid message Figure 5.4 Cleaner quick connect error message 36 CyAn ADP with Summit Software User Guide

The LED indicators on the front panel of the Sheath Management System also change when fluid levels change in the SMS. Three fluid levels are monitored on the SMS panel: SHEATH LEVEL, CLEANER LEVEL, and WASTE LEVEL. When all fluids are at suitable levels for proper operation of the CyAn ADP, the SHEATH LEVEL, CLEANER LEVEL, and WASTE LEVEL LEDs appear green beside the OK label: When the sheath level or cleaner fluid level is low, the green OK LED changes to a flashing amber or a solid amber LED beside the LOW label: When the waste fluid level is high, the green OK LED changes to a flashing amber LED beside the HIGH label: CyAn ADP with Summit Software User Guide 37

When the sheath or cleaner level changes from low to empty, the flashing amber LED changes to a red LED beside the EMPTY label: When the waste fluid level changes from high to full, the flashing amber LED changes to a red LED beside the FULL label: 38 CyAn ADP with Summit Software User Guide

Table 5.1 lists the indicators in the CyAn Control Panel user interface and the message text for each error or status condition. Table 5.1: CyAn ADP Control Panel Indicators CyAn Control Panel Message Text Message 102: Your CyAn cover is open. The laser shutters have been closed and fluidic system is shutdown. Please close your cover and restart. Message 301: Cleaner fluid is low. Please replace the cleaner solution and press the Startup button on the CyAn Control Panel. Message 302: Cleaner fluid is empty. Please replace the cleaner solution and press the Startup button on the CyAn Control Panel. Message 304: Cleaner quick connect is not completely engaged. Please check your connection. Message 305: Internal reservoir overfilled. This will not prevent operation of the instrument but may require future service. Message 306: Clean subsystem is halted. Please check that you have sufficient cleaner fluid. Message 307: Cleaner subsystem switch error. This will not prevent operation of the instrument but may require future service. Message 430: Less than 30 min of sheath fluid is remaining. Message 410: Less than 10 minutes of sheath fluid is remaining. Please replenish your sheath fluid. Message 401: Internal sheath reservoir level is low. CyAn will stop soon. Please replenish your sheath fluid. CyAn ADP with Summit Software User Guide 39

CyAn Control Panel Message Text Message 402: Out of sheath fluid. Replenish sheath and press the Startup button on the CyAn Control Panel. Message 404: Sheath quick connect is not completely engaged. Please check your connection. Message 405: Internal reservoir overfilled. This will not prevent operation of the instrument but may require future service. Message 406: Sheath subsystem is halted. Please check that you have sufficient sheath fluid. Message 407: Sheath subsystem switch error. Service maybe required. Message 705: Waste subsystem is halted. Please check waste tank level. Message 730: Less than 30 min until the waste container is full. Message 710: Less than 10 min until the waste container is full. Please empty the waste container. Message 700: Waste container is full. Please empty the waste container. Message 905: Low sheath pressure inside CyAn. Check connection between the CyAn and SMS. Message 906: Low vacuum inside CyAn. Check connection between CyAn and SMS. Message 997: Low sheath pressure in SMS. 40 CyAn ADP with Summit Software User Guide

CyAn Control Panel Message Text Message 998: Waste subsystem halted due to loss of vacuum. If during operation, please check vacuum pump, waste quick connect or waste tank level. Check the waste filter on the waste container to see if it has clogged. If so, replace the waste filter. <Message in top portion of Control Panel> Message 999: SMS Power fault. Reset SMS to continue. If this problem reoccurs, please call Dako for technical support. Changing Out Sheath and Waste Containers Use the shutdown fluidics procedure to fill and empty sheath and waste containers when running samples. 1. Click Fluidics Off on the CyAn Control Panel. WARNING Remove the waste container and the sheath container from the cart before emptying or filling. Dispose of the contents of the waste container in accordance with local, state, and federal regulations. Do not drop the waste or sheath containers on the Sheath Management System. Doing so may result in improper calibration of the load cells. 2. At the waste container, release the quick-connects. 3. Remove the waste container from the cart, remove the lid, and empty. Dispose of the contents of the waste container in accordance with local, state, and federal regulations. 4. Place an appropriate amount and type of disinfectant in the bottom of the waste tank to ensure effective killing action when the tank is full. All biologicals introduced into the waste tank must be in contact with the disinfectant for a minimum of 10 minutes before being discarded. See Appendix A on page Error! Bookmark not defined. for a list of Approved Cleaners and Disinfectant products. As an example: If 200mL of regular household bleach (5.25% active chlorine) is placed in the container, this will provide approximately 500ppm available chlorine when the tank s full capacity of 20L has been reached. If the samples you are running will not be effectively killed by this concentration of sodium hypochlorite solution, an increased amount of bleach should be used to achieve effective disinfection concentration. Please keep in mind that diluted sodium hypochlorite solutions begin losing their effectiveness after 24 hours. 5. Replace the lid and tighten, place the container back in its position on the cart, and reconnect the waste container using the quick-connects. CyAn ADP with Summit Software User Guide 41

6. Restore the sheath fluid by either replacing the entire sheath cubitainer or refilling the plastic carboy, depending on which type of sheath container is used with your Sheath Management System: If you are using the disposable sheath cubitainer, release the quick-connects. Dispose of the entire cubitainer in accordance with local, state, and federal regulations (for example, recycle the outside cardboard box and place the plastic bladder in an appropriate receptacle). Place a new sheath cubitainer back in its position on the cart. Locate the quick connect inside the box. Spray the quick connect with 70% ethanol. Reconnect the cubitainer to the SMS cart. If you are using the plastic carboy, release the quick-connects. Remove the carboy from the cart, loosen the lid, and fill with particulate-free deionized water (dh2o) or a suitable sheath fluid. Seal the lid, and place the container back in its position on the cart. Spray the quick connect with 70% ethanol. Reconnect the sheath container to the SMS cart using the quick-connects. 7. Click Startup on the Cyan Control Panel. Replacing Cleaner Fluid 1. Release the cleaner cubitainer quick-connect. 2. Unscrew the cap from the spent cleaner cubitainer. Retain this cap for use on the new cubitainer. Do not place this cap and its attached tubing on the floor or other surface where it will be in contact with contaminants. Place on clean paper towel if needed during change out of the cleaner cubitainers. 3. Dispose of the entire cleaner cubitainer in accordance with local, state, and federal regulations. (for example, recycle the outside cardboard box and place the plastic bladder in an appropriate receptacle). 4. Remove the cardboard punch-out on a new cleaner cubitainer. Holding onto the ring around the cap, pull up on the lid so that the lid extends up to the cardboard. 5. Remove the cap and replace it with the cap from the previous cubitainer. 6. Place the new cleaner cubitainer back in its position on the cart. Spray the quick connect with 70% ethanol. Reconnect the cubitainer to the SMS cart. 42 CyAn ADP with Summit Software User Guide

Section 6 Summit Software Basics Acquire and Analyze up to 10 Million Events Summit software, on CyAn ADP with all parameters enabled, has the capability to acquire, save, and analyze up to 10 million events. The following graphics depict 10 million events acquired at 25,000 events per second, during an eight color experiment, with 37 parameters enabled on the CyAn ADP. CyAn ADP with Summit Software User Guide 43

The following procedures describe some of the most commonly-used functions of Summit software. For complete Summit software documentation, see the Summit online help by clicking Table of Contents on the Summit Help menu. Create a New Database 1. Double-click the Summit software shortcut on the Windows desktop. The Select database dialog box appears. 2. Select CYAN for the database from the drop-down list. This allows you to create a database for CyAn. 3. Click New. The Create Database dialog box appears. 44 CyAn ADP with Summit Software User Guide

4. Specify the folder to create the new database in by clicking the Save in drop-down list and navigating to the desired folder location. To create a new folder, click the Create New Folder button, type a folder name, and then press ENTER. 5. Specify a name for the database in the File name field, and then click Save. The new database is created and saved to the specified folder. Immediately following this procedure, Summit software will open the newly-created database when launched. CyAn ADP with Summit Software User Guide 45

Open an Existing Database 1. Double-click the Summit shortcut on the Windows desktop. The Select database dialog box appears. 2. Click the Browse button. The Open Database dialog box appears. 3. Find the database you want to open by using the Look in drop-down list to browse all local drives and folders. 4. Select the database file you want to open and click Open. 46 CyAn ADP with Summit Software User Guide

Summit software now opens using the selected database. Create a Histogram 1. Select the Histogram tab in the Control Panel. 2. In the Create Histograms panel, double-click a parameter for the X Axis in the X-parameter column. CyAn ADP with Summit Software User Guide 47

A histogram is created with the selected parameter. Create a Dot Plot 1. Select a parameter for the X Axis in the X-parameter column. 2. Double-click a parameter for the Y Axis in the Y-parameter column. 48 CyAn ADP with Summit Software User Guide

A dot plot is created with the selected X and Y Axis parameters. Open FCS (Listmode) Files When a data file is opened in Summit software, the entire data set is not fully copied into the current database. Instead, a link or shortcut to the original file is created. A full listing of all opened files associated with the database can be viewed in the Database samples dialog box. 1. On the File menu, click Load Sample. The Open dialog box appears. CyAn ADP with Summit Software User Guide 49

2. Locate the FCS file to open by browsing to a folder in the Look in list. 3. Select the FCS file you want to open. To see more detailed information about the file, click More Info. 4. To select multiple files, press the CTRL key while selecting the files. 5. Click Open. The selected FCS file(s) are opened in Summit software. 50 CyAn ADP with Summit Software User Guide

Set a Gate from a Single Region 1. Open a dot plot and create a region (R1) for gating. 2. Right-click in the region and then click Set Gates. CyAn ADP with Summit Software User Guide 51

3. Double-click in a target dot plot or histogram to apply the gate. Set a Serial Gate 1. Open a dot plot gated on region (R1). 2. Create a region (R2) in the dot plot. 52 CyAn ADP with Summit Software User Guide

3. Right-click in region (R2) and then click Set Gates. The Combine region and gate? dialog box appears. 4. Click Yes to activate serial gating. Click No to gate using only the specified region. 5. Check the Make my choice the default and don t ask me again check box to disable this dialog box in the future. If checked, this option is set as the default in Summit software. The default can be changed in the Preferences dialog box. CyAn ADP with Summit Software User Guide 53

6. Click in a histogram or dot plot to apply the gate. If you clicked Yes in step 4, the serial (or sequential) gate logic is applied to the histogram or dot plot. The graphic below shows a gated histogram involving regions 1 and 2. Note When a region in a dot plot or histogram is used for gating, the serial gate option works by automatically appending any gate regions applied to that plot to the new gate. For example, a dot plot is gated based on two regions (R1 & R2). If an R4 region is created in that plot and is used as a gate on another histogram or dot plot, the serial gate option defines the newly applied gate as (R1 & R2 & R4). Electing not to serial gate would apply a gate involving region (R4) only to the target histogram or dot plot. 54 CyAn ADP with Summit Software User Guide

Define Gate Logic 1. Select the Gating tab in Control Panel. 2. Click the upper-left icon in the Gate Logic Builder panel and then click New to create a new gate. 3. Double-click a text field in the Expression column. The Edit gate expression dialog box appears. CyAn ADP with Summit Software User Guide 55

4. Select one or more regions for the gate and then click OK. Gates can be defined to include those events that fall inside or outside specific regions. To clear all selected regions, click Clear. Note The number of regions available correlates to the number of regions created in the current workspace. The newly defined gate expression is displayed in the Expression column of the Gate Logic Builder column and the current gate scheme is displayed in the Gate Scheme panel. 56 CyAn ADP with Summit Software User Guide

Shortcut Keys The following list describes available keyboard shortcuts in Summit software. These shortcuts are provided to help you quickly perform various operations in the software. Shortcut Key F1 F2 F3 F7 F8 Left arrow key Right arrow key Function Opens the Summit software online help system. Starts/Stops acquiring events. Saves acquisition data to a drive (C:, D:, etc.), the network, disk, or CD. Opens the Create New Histograms dialog box for the current workspace. Opens a window that displays all loaded sample files in the current database. Sets the displayed sample in the current workspace or protocol as the first file in the Database samples window. If more than one file group is present in the sample window (that is, organized in multiple folders), the last sample in the currently selected group is displayed. Also repositions a region to the left when selected. Sets the displayed sample in the current workspace or protocol as the last file in the Database samples window. If more than one file group is present in the sample window (that is, organized in multiple folders), the last sample in the currently selected group is displayed. Also repositions a region to the right when selected. Down arrow key Sets the displayed sample in the current workspace or protocol as the next file in the Database samples window. If more than one file group is present in the sample window (that is, organized in multiple folders), the last sample in the currently selected group is displayed. Also repositions a region downward when selected. Up arrow key Sets the displayed sample in the current workspace or protocol as the previous file in the Database samples window. If more than one file group is present in the sample window (that is, organized in multiple folders), the last sample in the currently selected group is displayed. Also repositions a region upward when selected. SHIFT+ARROW Expands a region in the arrow direction (left, right, up, down) when selected. CTRL+ARROW CTRL+O CTRL+S CTRL+P CTRL+C CTRL+V CTRL+D Contracts a region in the arrow direction (left, right, up, down) when selected Opens a dialog box to open one or more FCS listmode files. Saves modified items on the Summit desktop that have changed (or auto-save is invoked at periodic intervals and when Summit software is closed). Prints the current view of the Summit software desktop. Copies the selected region, which can be pasted into a histogram. Pastes the copied region into a histogram. Opens the Edit Gate Expression dialog box. CyAn ADP with Summit Software User Guide 57

Shortcut Key CTRL+Z ALT+F4 Function Clears the event buffer of all acquired events. Exits Summit software. + Expands one node when viewing a folder list in a window (use + on the numeric keypad). - Collapses one node when viewing a folder list in a window (use on the numeric keypad). * Expands an entire folder tree contained in a window (use * on the numeric keypad). 58 CyAn ADP with Summit Software User Guide

Section 7 CyAn ADP Maintenance Regular maintenance of the CyAn ADP instrument is recommended as described in this section. System integrity can be effectively maintained by assuring cleanliness of the fluidics. In addition to performing preventive maintenance procedures, we also recommend that you establish and perform other laboratory procedures for routine operations such as backing up your data and experimental protocols. Daily Preventive Maintenance Follow the cleaning steps outlined in Section 4: Startup and Shutdown Procedures on page 32 on a daily basis. Weekly Preventive Maintenance Reusable Sheath Containers The following steps should be performed on a weekly basis. If using the reusable sheath carboy, it should be cleaned and disinfected once a week. The container should be handled as aseptically as possible during cleaning. A location where a sink, hot water, counter space, and DI water are available is required. 1. Put on gloves, a lab coat, and safety glasses. 2. Remove cap of container and set aside on clean paper towel. 3. Add a small amount of a phosphate-free laboratory grade soap and enough hot water to create a lather. Replace cap. Agitate or shake vigorously for 30 seconds. 4. Empty out soap and rinse with hot water until soap is removed. 5. Rinse with DI Water. 6. With 70% Ethanol, spray the cap and threads on the container and the exterior of container and wipe clean. 7. The carboy can now be filled with sheath fluid. Note: If using the disposable sheath cubitainers, no weekly maintenance of the sheath container is needed; however, please follow the instructions starting on page 41 when switching out disposable sheath cubitainers. CyAn ADP with Summit Software User Guide 59

Head Unit and SMS Cart The exterior surfaces of the CyAn ADP should be cleaned on a weekly basis. Any generalpurpose surface cleaner/disinfectant can be used. All surfaces should be wiped down to remove dust, fingerprints, and any other soiled marks. The sample injection area should be thoroughly cleaned and disinfected as well. It is highly recommended that the computer keyboard and especially the mouse are cleaned and disinfected on a weekly basis also. Fluidic Lines SMS Clean & Rinse Cycle Note: Before running the clean & rinse cycle, make sure you have enough sheath fluid and cleaner fluid to complete the cycle. On the CyAn Control Panel, click the Clean/ Rinse button. A message box will warn you that the Clean and Rinse process will take 10-15 minutes. Click OK to continue. The clean cycle takes seven minutes to complete, followed by a rinse cycle that takes seven minutes to complete. Monthly Preventive Maintenance System Decontamination Performing this decontamination procedure is a necessary part of system maintenance and will ensure the instrument s longevity. To prevent the buildup of debris and potential contamination of the fluidics system on the CyAn ADP, the system must be decontaminated on a monthly basis. A disinfectant should be run through the system to eliminate any sample buildup or microorganisms that may have entered the system through normal everyday use. Only Dako-approved disinfectants can be used. The following procedure must be followed once per month. A reusable sheath carboy is required. 1. Ensure that the instrument is ON and the software is open with at least one histogram created. 2. Put on gloves, safety glasses, and a laboratory coat. Shut down the fluidics on the CyAn ADP. 3. In a clean reusable sheath carboy make a minimum of two liters of a 1:5 dilution of Dako Decontamination Solution (equivalent to 2000 ppm active chlorine). 4. Spray the quick connect with 70% Ethanol and hook up to sheath port on the SMS cart.* 5. Start up the fluidics. This will move the Decontamination Solution in the sheath tank through the system. 6. Place a test tube with 4mL of 1:5 diluted Decontamination Solution on the sample probe. 7. Press F2 to acquire. Sample flow rate should be on LOW (corresponding to ~ 10-20 µl/min). 8. Let the Decontamination Solution run from both the sheath tank and the sample for 60 minutes. 60 CyAn ADP with Summit Software User Guide

9. After 60 minutes, click Debubble on the CyAn Control Panel. After this function is complete, remove the tube from the sample probe and Backflush once. 10. Shut down the fluidics on the CyAn ADP. 11. Replace sheath with DI water. Hook up a disposable box of sterile DI water or thoroughly rinse the reusable carboy with DI water and then fill with 5 liters of fresh DI water. 12. Spray quick connect with 70% Ethanol and hook up to sheath port on SMS cart.* 13. Start up fluidics and place a tube with 4mL DI water on the sample probe. 14. Click F2 to acquire. Sample flow rate should be on LOW. 15. Let rinse for a minimum of 90 minutes. Note: DI water may need to be refilled on the sample probe during the rinse time. Refill as necessary. 16. After 90 minutes have elapsed, click Debubble on the CyAn Control Panel. After this function is complete, remove the tube from the sample probe and Backflush once. 17. Perform an SMS Clean & Rinse Cycle. See Weekly Preventative Maintenance Fluidic Lines above. The system is now fully decontaminated and ready to continue cellular analysis. *Note: A sheath container with approximately 1L of fluid in it may alert the system that the tank is low and that it should be changed out soon. The decontamination cycle will be able to run for the specified time without the instrument fluidics stopping. Please call Customer Care if any problems arise with performing this procedure. Laser Interlock Maintenance Procedure 1. Follow the instrument startup procedure. Make sure the lasers are on and the laser shutters are open. 2. Visually inspect the housing on the instrument to verify that panels or covers are fitted and tight so that all laser energy is contained in the interior. 3. Facing the instrument, lift the lid approximately one inch (2.54 cm). The LED light(s) on the right front of the cover should go out, verifying that the laser shutters are closed. 4. Close the lid. 5. If the LED light(s) did not go out when the lid was opened, contact your local Dako Support Representative. Biannual Maintenance Procedure A Dako Field Service Representative should perform an maintenance check on the CyAn ADP every 6 months. To schedule a 6-month or annual maintenance service, contact your local Dako Support Representative. CyAn ADP with Summit Software User Guide 61

62 CyAn ADP with Summit Software User Guide

Section 8 Troubleshooting Please contact your local Dako Field Service Representative immediately for assistance with any instrument malfunction or service need. WARNING Do not attempt any maintenance on the CyAn ADP laser components. Laser maintenance should only be performed by specially trained, certified Dako Field Service Representatives. The following table is a guide for troubleshooting CyAn ADP problems. Table 6.1: CyAn ADP Troubleshooting Guide Problem CyAn ADP has no power Acquiring data, but no display. Median values spike or fall suddenly and recover on all or most fluorescence channels across multiple laser paths. Indicator lights in the Sheath Management System portion of the Control Panel change from green to amber or red. Data integrity data looks questionable during acquisition. Action Make sure that the power plug is firmly attached to the wall. Make sure that the power switch on the back of the CyAn ADP is on. There are no operatorreplaceable fuses in the CyAn ADP. Make sure that the lasers are on. Make sure that the laser shutters are open on the CyAn Control Panel. Debubble (Click Debubble on the Control Panel.) Hover the cursor over the indicator light to get more information about the error and instructions on how to correct the problem. Information on the problem can also be found in Section 5. Remove sample. Perform backflush and debubble. Check sample preparation. Re-run sample. If problem remains, check alignment with beads. Perform another backflush and debubble. If problem persists, contact Customer Care. CyAn ADP with Summit Software User Guide 63

64 CyAn ADP with Summit Software User Guide

Section 9 Six-Color Compensation Experiment Background Compensation is the process of resolving the actual intensities from each antibody conjugate in a multicolored sample. Using single color staining or single color fluorochromes as a compensation control makes it possible to define the relative amount of light that ends up in another detector. For example, compensation can be used to determine how much light from FITC ends up in the FL2 detector and, conversely, how much light from PE ends up in the FL1 detector (see figure below). This percentage is defined as the spillover coefficient of FITC into FL2 and PE into FL1. By having these spillover coefficients defined for all colors and channels in an experiment, it is possible to calculate the compensation of data in a multicolor sample using linear algebra. This is the way that compensation is performed mathematically in Summit software and other offline flow analysis software. The amount of light that is detected in each fluorescence parameter (FL1, FL2) is the area under the curve of each spectra. If two fluorochromes are bound to the same cell, the sum of the light from each fluorochrome is detected. Knowing the spillover in both directions makes it possible to mathematically determine the light from each fluorochrome. For two fluorochromes, the following math is used: FL1(FITC) = FL1(total) a 12 FL2(total) FL2(PE) = FL2(total) a 21 FL1(total) where a 12 is the spillover of FITC into FL2 and a 21 is the spillover of PE into FL1. The (total) is the signal measured on the dual stained cell in each channel. This math can be extrapolated into an infinite number of colors. CyAn ADP with Summit Software User Guide 65

The following 6-color experiment utilizes the Blue (488nm), Violet (405nm) and Red (635nm) lasers, uses the fluorochromes FITC, PE, PE-Cy5, Cascade Yellow, Pacific Blue and APC, and involves compensation. Whole blood is drawn from a donor and partitioned into 8 tubes. Tube # 1 is not stained with any fluorochrome (a "no stain" control). Tubes # 2-7 are each stained using one of the 6 colors. Cells within tube # 8 are stained using all 6 fluorochromes. To the right is a table listing the 8 tubes, fluorochromes used for each, and the respective cell surface marker targeted. *Please note that in Tube # 6 we have used CD3 Cascade Yellow and not CD45 Cascade Yellow as added in Tube # 8. By using CD3 Cascade Yellow, an adequate number of positive and negative events will be seen, making compensation setup easier. In general, it is possible to use any fluorochrome antibody conjugate with the same fluorochromes to set compensation. If the lymphocyte gate (R1) is set correctly, then the number of CD45 negative events seen will be minimal, making derivation of compensation settings difficult. In Tube # 8 the combination of CD45 Cascade Yellow and Side Scatter can be used to set a more accurate lymphocyte gate by allowing for the exclusion of debris, basophils and monocytes. Setting up for sample acquisition When performing a multi-color experiment, the forward and side light scatter parameters should be set to linear; all fluorescent parameters should be set to log. The range of expression of various antigens, e.g. CD3, CD4, CD8 etc, on a cell can be very large. This is why we need to use a logarithmic scale to ensure we can see the whole range of antigen expression from dim to very bright. When staining for DNA we are usually only looking for a doubling in fluorescence thus a linear scale is preferred. Sample Target Stain Tube # 1 None None Tube # 2 CD4 FITC Tube # 3 CD56 PE Tube # 4 CD19 PE-Cy5 Tube # 5 CD8 APC Tube # 6 CD3* Cascade Yellow Tube # 7 CD3 Pacific Blue Tube # 8 CD4 FITC CD56 CD19 CD8 CD45* CD3 PE PE-Cy5 APC Cascade Yellow Pacific Blue 66 CyAn ADP with Summit Software User Guide

Create a forward vs. side scatter dot plot and run the non-stained sample (Tube # 1). This allows identification of the cell populations that will be used for gating. Below is a dot-plot showing unstained lysed and washed whole blood, using both the forward and side scatter parameters. The lymphocyte, monocyte, and granulocyte populations have been color coded for easy recognition. Within the forward-side scatter plot, create a tight region around the lymphocyte population (R1). This region will be used to gate the lymphocytes into additional histograms. Now make a single or dual parameter histogram in log for each color of interest gated on the lymphocyte from the FSC / SSC plot. Adjust the PMT voltage for each color so that the unstained lymphocyte population is situated within the first log decade for all colors. It is important the PMT voltage is set fairly high to prevent the median of the negative becoming 1, because that will add uncertainty to the manual compensation (see page 72). Acquire the single color compensation controls/tubes, as well as the multi color tube(s), with these PMT settings. CyAn ADP with Summit Software User Guide 67

Setting Up Histograms for Compensation When performing a multi-color experiment, a non-stained sample (Tube #1) is run, followed by the first "single color" stain to be analyzed (e.g. Tube # 2 FITC). Create a series of plots such that this parameter is paired with each of the other colors in the experiment. For example, this 6-color experiment involves FITC, PE, PE-Cy5, Cascade Yellow, Pacific Blue and APC. If FITC is the first "single color" to be run, 5 plots should be created: FITC vs. PE, FITC vs. PE-Cy5, FITC vs. APC, FITC vs. Cascade Yellow, and FITC vs. Pacific Blue. Typically, we recommend that you plot FITC along the X-axis and the other 5 colors along the Y. Additionally, you will need to create quadrant regions within each of the dot plots except FSC vs. SSC. Statistics from the quadrant regions will be used in setting compensation from the control samples and identifying positive and negative populations in the "all stains" sample. In general, we recommend that you position the quadrants to encompass the first 1.5 log decades. The graphic below shows 5 plots that have been created for the 6-color data. Notice that FITC has been paired against the other 5 fluorochromes used in the experiment. 68 CyAn ADP with Summit Software User Guide

After creating the necessary histograms and quadrant regions, apply the gate from the lymphocyte population region (R1) to the 5 dot plots. CyAn ADP with Summit Software User Guide 69

Load the non-stained sample Tube # 1 to verify that the PMT settings were adjusted correctly during acquisition. 70 CyAn ADP with Summit Software User Guide

Manual Compensation After gating the dot plots for the lymphocyte population within region (R1) in the FSC vs. SSC dot plot, load the first "single color" sample. In theory, it does not matter which of the 6 "single color" samples you begin with, although in this example we are starting with FITC. You will notice that in the FITC vs. PE plot, the positive population is shifted into the upper right quadrant. This means the signal from the FITC population is also being detected by the PE parameter. The degree to which signal is being detected for the PE parameter represents the % spillover of PE into the FITC signal. CyAn ADP with Summit Software User Guide 71

Setting Spillover Percentage for Compensation For proper compensation, adjust the spillover percentage so that the Y-axis median fluorescence value for the positive population (lower right hand quadrant) is equivalent to that seen with the negative population (lower left hand quadrant). A range of ± 1.0 is usually acceptable. By adjusting the spillover percentage value, the negative population might obtain a value of 1 before the median of the positive and negative population are aligned (thus the high PMT settings as previously explained). If so, you might need to adjust the spillover percentage without having the negative to align to, which can result in overcompensation. Overcompensation in general is to be avoided, as this can result in populations of interest disappearing when analyzing the multicolor samples. Negative cell population Y-axis median fluorescence value Positive cell population Y-axis median fluorescence value Lower left hand quadrant = Negative cell population Lower right hand quadrant = Positive cell population 72 CyAn ADP with Summit Software User Guide

Running the Second Sample Next, load the second "single color" sample. Before doing this, change the parameters of each of the histograms such that the second color (in this case PE) is plotted against the other 5 colors: PE vs. FITC, PE vs. PE-Cy5, PE vs. APC, PE vs. Cascade Yellow and PE vs. Pacific Blue. Two populations will be delineated, one being PE negative and the other positive. CyAn ADP with Summit Software User Guide 73

Again, compare the Y-axis median fluorescence between the lower left and lower right quadrants, and adjust compensation as needed. Why adjust compensation for the PE vs. FITC plot when FITC vs. PE was compensated for the FITC sample? This is because the % fluorescent spillover occurs both ways, FITC into the PE parameter and PE into the FITC parameter. Look at the spectral emission curves for both fluorochromes. You will notice that the degree of spillover between the two fluorochromes actually differs. This is why compensation must be adjusted based on both the FITC and PE "single color" samples. 74 CyAn ADP with Summit Software User Guide

Running the Remaining Samples Now run the third "single color" sample, in this case PE-Cy5. Again, remember to reset the parameters along the dot plot axes so that PE-Cy5 is plotted against the other 5 colors. Note, these steps will be repeated for the remaining "single color" samples. CyAn ADP with Summit Software User Guide 75

Where necessary, adjust compensation to align the Y-axis median fluorescence (relative to the other 5 colors) between the lower left and lower right quadrants. 76 CyAn ADP with Summit Software User Guide

Run the 4th sample (APC). CyAn ADP with Summit Software User Guide 77

Where necessary, adjust compensation to align the Y-axis median fluorescence (relative to the other 5 colors) between the lower left and lower right quadrants. 78 CyAn ADP with Summit Software User Guide

Run the 5th "single color" sample (Cascade Yellow). CyAn ADP with Summit Software User Guide 79

Where necessary, adjust compensation to align the Y-axis median fluorescence (relative to the other 5 colors) between the lower left and lower right quadrants. 80 CyAn ADP with Summit Software User Guide

Run the 6th "single color" sample (Pacific Blue). CyAn ADP with Summit Software User Guide 81

Where necessary, adjust compensation to align the Y-axis median fluorescence (relative to the other 5 colors) between the lower left and lower right quadrants. Now the spillover percentage is defined for all the combinations of colors and the multicolored sample can be analyzed. This experiment has demonstrated offline compensation; however, compensation can be applied during acquisition as well. 82 CyAn ADP with Summit Software User Guide

Running the All Stains Sample Finally, run the "all stains" sample. This sample can be analyzed using dot plots containing any combination of the 6 parameters: FITC, PE, PE-Cy5, APC, Cascade Yellow and Pacific Blue. If compensation has been properly set, the populations will be aligned so that the positive and negative populations can be easily identified. When performing Lymphocyte sub set analysis, it is preferable to use CD45 vs. SSC to locate the lymphocyte population than FSC vs. SSC. CD45 vs. SSC gating allows for the determination of a cleaner lymphocyte population by excluding other contaminating cell populations from the gate. The plots shown below illustrate this. If the lymphocyte gate is set using FSC vs. SSC it is likely to include some debris (Blue), basophils (Green) and some monocytes (Gray). These contaminating populations can be easily identified and excluded using a CD45 vs. SSC plot. Debris (Blue) Monocytes (Gray) Lymphocytes (Red) Basophils (Green) CyAn ADP with Summit Software User Guide 83

Create a plot showing CD45 (Cascade Yellow) vs. SSC and draw a region enclosing the lymphocyte population that have low SSC and bright CD45 staining, taking care to exclude the basophils and monocytes. Apply this gate to the rest of the plots. Putting Compensation into Practice This tutorial has demonstrated how to perform a 6-color experiment involving compensation. However, after running the "all stains" sample, how does one make sense of and use the compensated data from the "all stains" sample? The following histograms illustrate the different cell populations that can be identified, analyzed, and/or sorted based on the 6-color experiment. For each of the cell populations below, identification is based upon the presence or absence of specific cell surface markers. To recall which marker correlates to which fluorochrome; refer to the original tube setup table at right. Tube # 8 CD4 FITC CD56 CD19 CD8 CD45 CD3 PE PE-Cy5 APC Cascade Yellow Pacific Blue CD3+ T-Lymphocytes Create a dot plot showing compensated CD3 Pacific Blue vs. compensated CD19 PE-Cy5 gated through the lymphocyte gate R1. The CD3+ T-Lymphocyte population appears in the lower right hand quadrant (R42 in the example shown). So in this case the CD3+ T-Lymphocyte population constitutes 69% of the total lymphocyte population. CD 19+ B-Lymphocytes Using the compensated CD3 vs. compensated CD19 plot, the CD19+ B-Lymphocytes are found in the upper left quadrant (R39). 84 CyAn ADP with Summit Software User Guide

CD4+/CD3+ T-Lymphocytes (T-Helper cells) Create a dot plot showing compensated CD3 Pacific Blue vs. compensated CD4 FITC. The CD4+/CD3+ T-Lymphocytes are found in the upper right quadrant (R44). Events found in the upper left quadrant (R43) that are CD4 dim and CD3 negative are contaminating monocytes in the lymphocyte gate. CD8+/CD3+ T-Lymphocytes (T-Cytotoxic/Suppressor cells) Create a dot plot showing compensated CD3 Pacific Blue vs. compensated CD8 APC. The CD8+/CD3+ T-Lymphocytes are found in the upper right quadrant (R36). Events found in the upper left quadrant (R35) that are CD8 dim and CD3 negative are sub set of Natural Killer lymphocytes. CyAn ADP with Summit Software User Guide 85

Natural Killer Lymphocytes Create a dot plot of compensated CD3 Pacific Blue vs. compensated CD56 PE. Natural Killer lymphocytes are found in the upper left quadrant (R31). Occasionally a small population of T-Lymphocytes can be seen which also express CD56. These Natural killer like lymphocytes can be distinguished from true Natural Killer lymphocytes because they are also positive for CD3. (See upper right quadrant R32). 86 CyAn ADP with Summit Software User Guide

Section 10 Auto Compensation Summit software provides an automatic method to obtain a full compensation matrix for multicolor analysis. The compensation matrix is calculated from single stained controls by the autocompensate function. Auto Compensation for Single Positive Controls The following procedure describes how to use Summit software Auto Compensation feature within single stained controls. Although this feature provides an automatic method to compensate data, compensation can still be adjusted, fine-tuned, or performed manually or via the compensation matrix. 1. Acquire the single-control samples required for your experiment. The first control sample should include an unstained or isotype control for which you will set PMT Voltages. From the resulting dot plot, you can determine gating if required. Any gates that you want to use must be set before you apply Auto Compensation. Note: During the Auto Compensation operation, adjustments to only the size and placement of regions are allowed. 2. Run the remaining single control samples and save the data files. 3. Load all control sample files into an experiment folder. 4. Click the Sample tab. 5. In the Sample Compensation panel, click the small, blue icon in the upper-left corner and select Auto Compensate from the list. CyAn ADP with Summit Software User Guide 87

The Auto Comp Sample dialog box appears. 6. Select a gate from the Gate list, if applicable. 7. From the Experiment list, select the experiment folder that contains your control samples. 8. Select all of the single control samples included in the experiment. 9. Click OK. A new Workspace labeled AutoComp is created and the first set of dot plots is displayed (see figure 10.1). Each dot plot places the control parameter on the x-axis and a parameter to compensate against on the y-axis. Default auto compensation Dim and Bright regions are displayed and, if a gate was selected, it is applied to each dot plot. The Auto Compensate dialog box appears. Important If you click Cancel at any point in the auto compensation process, you will clear the compensation matrix and the AutoComp workspace. 88 CyAn ADP with Summit Software User Guide

Figure 10.1 Single Control Sample Dot Plots 10. Examine the % Hist statistics for each histogram. If either the Dim or Bright region contains less than 5% of the data for the dot plot, click-and-drag the region until greater than 5% of the data appears in both the Dim and Bright regions. CyAn ADP with Summit Software User Guide 89

11. When all regions on all plots contain greater than 5% of the data, Click Next on the Auto Compensate dialog box. The next set of dot plots will appear. 12. Repeat step 10 until all single-control samples have been compensated. When auto compensation is complete, the compensation matrix contains the appropriate values and the AutoComp workspace is removed. 90 CyAn ADP with Summit Software User Guide

Section 11 VisiComp Using the VisiComp Feature after Compensation Prior to the development of Summit software version 4.3, compensation was done through logarithmic plots, which could not display 0 or negative values. Because these values were not displayed, you might have inadvertently over-compensated when compensating manually, or found it was difficult to verify the results of the auto compensation feature. To help you better visualize the results of compensation, Summit software now includes a new VisiComp scaling algorithm that displays 0 and negative values. VisiComp provides a good way to verify the results of the Summit software Auto Compensation feature, and allows you to fine tune and make adjustments to compensation. Recommended use of VisiComp: 1. Pre-load all necessary samples (Listmode *.fcs files) that are required to perform compensation. 2. Create all necessary plots, regions, and gates to establish the compensation gating. 3. Enable VisiComp. On the Sample tab, in the Compensation panel, click the icon select VisiComp. in the upper left, and 4. Use either the auto compensation wizard to set up plots, or manually set up all of the plots that you want to use for compensation analysis. 5. If you did not use the auto compensation wizard, perform auto compensation on a per-plot basis. CyAn ADP with Summit Software User Guide 91

6. To adjust the width of the VisiComp linear region, click the small, blue icon in the upper left portion of the dot plot and select Adjust VisiComp. Use the slider tool, or enter a specific value to complete the adjustment. Note: The adjusted width of the VisiComp linear region applies to all plots and histograms that display compensated parameters in any one sample template. Because of this, it is important to display all data before you adjust the width. What is ideal for one parameter pair may not be ideal for another. Therefore, adjust the width to display the best compromise across all plots. 7. Create regions and gates to complete your analysis. Note: When you make width adjustments after you have created regions, the software attempts to adjust the regions to the new scale. However, the result is not always precise due to cumulative rounding errors. Therefore, you must verify the locations of your regions if you adjust the width after you have created regions. Important: If you turn off VisiComp, any regions that extend into the negative area of the VisiComp scale will be moved where they can be displayed on the log scale. Any regions that were entirely in the negative area will have a 0 width and 0 height. 92 CyAn ADP with Summit Software User Guide

Section 12 WorkList Builder Overview WorkList Builder in Summit software allows you to create reagent and specimen/researcher databases and lists of tests to be run on Dako flow cytometers. Once your common panels and tests (tubes or samples) are defined, they can be easily incorporated into Worklists for use during acquisition. This guide demonstrates how to set up and define your panels tests (samples or tubes), and specify antibody-fluorochrome (Ab/FL) combinations, in addition to setting up Worklists. Included in this section is a setup for a six-color panel involving an unstained control, six singlecolor controls, and an All Stains sample. For purposes of instruction, this guide will follow the workflow below. 1) Accessing the WorkList Builder. 2) Creating and naming a panel. 3) Defining reagents (Ab/FL): you need these when creating tubes. 4) Creating tests (tubes or samples) and associating them with the correct reagents: you need these defined in order to incorporate them into panels. 5) Specifying the tests (tubes or samples) within a panel. 6) Setting up and creating Worklists from your defined panels and tests. Important: All examples in this section require the following Runtime Option to be turned off. The Disable unused parameters checkbox is clear by default. For most applications Dako recommends that you leave this off. Note: This option is only available when you are running WorkList Builder through Summit software online (Edit > Runtime Options). CyAn ADP with Summit Software User Guide 93

Accessing the WorkList Builder The WorkList Builder can be accessed directly within Summit software, either through the main Summit menus (View > Worklist Panel) or by selecting Ctrl + W. Alternatively, the WorkList Builder can be run independently of Summit software by selecting the WorkList Builder v2.0 icon on the desktop. 94 CyAn ADP with Summit Software User Guide

Creating a New Panel 1. The quickest and easiest way to set up panels is by using the Panel Wizard option. Click the Panel Wizard button to start the wizard. 2. A dialog box appears that asks you to create a new panel or edit an existing panel. Use the radio buttons to make a selection and then press the Next button to continue. CyAn ADP with Summit Software User Guide 95

3. Another dialog box appears where you can enter the name and a description of your panel. The next step is to define the tests (tubes or samples) that comprise your panel and designate the reagents and antibodies used for staining. 4. Click the New Test button within the dialog box to set up and define the tests (tubes or samples) within your panel. 96 CyAn ADP with Summit Software User Guide

Setting up Tests and Defining Reagents for a Panel The Create New Test dialog box appears. This box enables you to define individual tests (tubes or samples) within a panel and specify the Ab/FL combinations within each. If you are creating and setting up tests (tubes or samples) for the first time, you will first need to define the reagents and staining combinations for the individual tests. In the example below, you can see that no reagents are listed within the Available Reagents field. 1. To define your reagents, click the New Reagent button. CyAn ADP with Summit Software User Guide 97

Creating Reagents by Defining Antibody/Fluorochrome Combinations The Create New Reagent dialog box appears. Here you can specify the Ab/FL combinations for your experiment. 1. In the top-left drop-down box type in a name for the new reagent. Make sure this label is descriptive of the Ab/FL combination. The example below assumes the experiment includes the CD4/FITC Ab/FL combination as the reagent. The reagent name you specify will also be displayed within the Create New Test dialog box, where you will define your tests within the panel. Reagent names are listed alphabetically. 2. Select the antibodies and fluorochromes associated with the new reagent. By default, the reagents from the drop-down boxes will be used for the label that will appear alongside your histogram and dot plot axes when the sample is run. Additional options exist where you can enter the clone, lot number, product code, description, etc. for the reagent. 3. Once you have defined the reagent by entering the Ab/FL combination, and have entered all other relevant information, select the Add Ingredient button. Reagent information that you entered should now appear in the Bead/Reagent Ingredients field. 4. Click the Save and Close button to add the reagent to the Create New Test dialog box. 98 CyAn ADP with Summit Software User Guide

5. Repeat this process until you have defined all the Ab/FL combinations that are routinely used in your panels or experiments. Note: A reagent database is provided with the WorkList Builder which contains all of the Ab/FL combinations provided by Dako. Using this database will reduce the need to set up and define your own reagent combinations. 6. When you are finished, click the Save and Close button to return to the Create New Test dialog. You will see all of your defined Ab/FL combinations displayed in the Available Reagents field. CyAn ADP with Summit Software User Guide 99

Deleting Reagents from the WorkList Builder Database 1. Select Edit > Edit Reagents from the main WorkList Builder menu. 2. The Reagents Database Editor appears. Use the drop-down box in the upper-left corner to select a reagent to delete. 3. Click Delete Reagent. 4. Click Clear All Fields. 5. Click Close Editor to exit the dialog box. Note: When deleting a reagent that has previously been incorporated into a panel(s), the associated panel(s) will also be deleted. This means that the panels will no longer be available for use within Worklists unless they are recreated. 100 CyAn ADP with Summit Software User Guide

Setting up Tests (Tubes or Samples) within a Panel Next you will define the individual tests (tubes or samples) within your panel and attach the corresponding Ab/FL combination to the tests. The following example explains how to set up a six-color panel that consists of eight tubes. Tube 1: Unstained control Tube 2: Single color CD4/FITC control Tube 3: Single color CD56/RPE control Tube 4: Single color CD19/RPE-Cy5 control Tube 5: Single color CD8/APC control Tube 6: Single color CD3/Pacific Blue control Tube 7: Single color CD45/Cascade Yellow control Tube 8: An All Stains sample (which contains each of the 6 Ab/FL combinations) Important: All examples in this section require the following Runtime Option to be turned off. The Disable unused parameters checkbox is clear by default. For most applications Dako recommends that you leave this off. Note: This option is only available when you are running WorkList Builder through Summit software online (Edit > Runtime Options). 1. Click Panel Wizard > Next > New Test to access the Create New Test dialog box. 2. The unstained control test is defined first. Enter a name for the unstained control test in the upper-left field on the screen. 3. Although you are setting up a test for the unstained control, you must specify all the fluorochromes that will be used to analyze the All Stains tube. Double-click in the Parameters in Test area in the lower right portion of the screen. Select a Fluorochrome and enter a Parameter Label. It is not necessary to enter an antibody. Click OK. Repeat until all fluorochromes in the experiment are represented. CyAn ADP with Summit Software User Guide 101

Note: Instead of creating a separate unstained control for each panel, you can alternatively create one unstained control test that includes all parameters. 4. Click Save Test, and click Close to add the test to your panel. 102 CyAn ADP with Summit Software User Guide

To set up the first single-color control test for FITC: 5. Return to the Create New Test dialog box. Enter a name for the sample (eg, Single Color FITC ) and select the appropriate reagent(s) for the test (eg, CD4/FITC). 6. Click Add Reagent to associate the reagent with the test. 7. Click Save and Close to add the test to your panel. CyAn ADP with Summit Software User Guide 103

8. For tests (tubes or samples) within your panel that contain multiple Ab/FL combinations, you must add all the relevant reagents. The picture below shows that all six reagents are added to the All Stains test. The last five reagents added are from the Dako reagent database that is automatically supplied when the WorkList Builder is installed. 9. Click the Save and Close button. 104 CyAn ADP with Summit Software User Guide

Setting up a Panel 1. Exit Create New Test and return to the main Panel set up dialog box. All of your defined tests with the corresponding Ab/FL combinations are displayed in this dialog box. 2. Click the Add button to specify which tests will be included in the Panel. 3. Click Next to continue. CyAn ADP with Summit Software User Guide 105

4. A final dialog box appears where you can verify that your panel-test-reagent information is correct. 5. Click Finish to create the panel and return to the main WorkList dialog box. 106 CyAn ADP with Summit Software User Guide

Creating Worklists from Defined Panels and Tubes 1. Return to the main Worklist dialog box. You will now see your panel and the individual tubes listed within Panels/Tests. Once panel(s) and tests are defined, it is easy to incorporate them into individual Worklists. 2. To create a Worklist, locate the Panels/Tests list and double-click the selection or selections you want to include. You can also highlight a panel or test and press Add to Worklist. All tests (tubes or samples) that make up the panel will be added to the Worklist as shown below. 3. Repeat the procedure to add any combination of panels and/or tests to a Worklist. 4. For information on how to run a Worklist, see the online Help in Summit software. CyAn ADP with Summit Software User Guide 107

Setting up Worklists for Multiple Experiments or Specimens The WorkList Builder also provides the capability to define and couple experiments and/or specify specimens for individual Worklists. 1. To set up and run a panel on two different specimens, double click and add specimens within the appropriate field. 2. New specimens will be labeled as Specimen 1, Specimen 2, etc., by default. However, specimens may be renamed by double-clicking on the Specimens label and entering a new name such as Mouse 1 and Mouse 2. 108 CyAn ADP with Summit Software User Guide

3. To set up a Worklist involving a six-color panel for both specimens, press the CRTL key or SHIFT key and select both specimens using the mouse. 4. Select the panel and/or tests to be included and click the Add to Worklist button. Once selected, a Worklist of the six-color panel will be created for both specimens (eg, Mouse 1 and Mouse 2). CyAn ADP with Summit Software User Guide 109

Customizing the WorkList Builder Viewing Area 1. In the main Worklist area, click the small, blue icon in the upper-left corner of the screen. The Show/Hide Columns option allows you to customize displayed information appearing next to the tests within a Worklist. Select Show/Hide Columns from the menu to access the Edit columns dialog box. Edit Columns Edit columns lists all the categories for your Worklist tests. Individual columns may be turned on or off through the checkboxes. You may also use the cursor to select and reorder items within the list. Columns within the Worklist areas are arranged according to the order of the items within Edit columns. 110 CyAn ADP with Summit Software User Guide

Importing a Specimen List from a Text File The WorkList Builder provides the capability to import a specimen list directly from a text file. 1. Create a text file as shown below and enter the specimen names. 2. Within the WorkList Builder main menu, select File>Import>Specimens. 3. Enter each specimen name on a separate line within the text file, or use the TAB key to separate each specimen name without hitting the ENTER key. CyAn ADP with Summit Software User Guide 111

Saving and Reusing Worklists Saving and opening Worklists can be performed through the main WorkList Builder menu. 1. To save a Worklist select File>Save As> Worklist from the main menu. 2. To open and use a previously created Worklist, select File>Open>Worklist from the main menu. 112 CyAn ADP with Summit Software User Guide

Associating Protocols, Gate/Event Limits, or Other Information to Worklist Tests The test Properties section provides a number of fields where descriptions, sample information, protocols, or stop conditions may be assigned to tests within a Worklist. 1. Double-click in any of the fields to add descriptions or information to the test, assign a protocol, or define stop conditions. If you want to associate a protocol with more than one test, press the CRTL key or SHIFT key and select the tests using the mouse. Note: A protocol must first be associated with a test in order for you to specify test templates or define gate or gate event information. 2. Double-click in the column field to the right of Protocol (see cursor) to access a dialog box where you can open, and associate a protocol, with the test and/or panel. CyAn ADP with Summit Software User Guide 113

3. The Define Summit Protocol dialog box appears. In this box you can associate a protocol to the test. By associating a protocol with a Worklist, the protocol will be automatically opened and used during acquisition within Summit software. For information on how to run a Worklist, see Summit software online Help. Note: Within this dialog box are two buttons allowing you to embed your protocol within the Worklist or simply reference the protocol. The Embed option creates and saves a full copy of the protocol within the Worklist, while the Reference option simply links a Worklist to the referenced protocol. Although embedding a protocol does slightly increase the size of a Worklist, this option is ideal for situations when the original protocol is moved, deleted, or unavailable. 114 CyAn ADP with Summit Software User Guide

Appendix A Approved Cleaners and Disinfectants The following list of cleaners and disinfectants can be used on the CyAn ADP. If products not specified on this list are used, it may cause damage to the system and void the warranty. Any questions or concerns regarding chemical usage on the CyAn ADP should be directed to Dako Customer Care. Cleaners 1. Dako Clean and Rinse Solution 2. 0.1% Triton-X100 in DI water 3. 70% Ethanol in DI water Disinfectants for Use in Sample Line* 1. Dako Decontamination Solution (undiluted, diluted 1:2, 1:5, or 1:10 depending on pathogenicity of sample) 2. 70% Ethanol in DI water *ANY disinfectant used on the sample probe must be rinsed with an equal amount of DI water. Disinfectants for Use in Sheath Line** 1. Dako Decontamination Solution (diluted 1:5) **ANY disinfectant used in the sheath lines must be rinsed with DI water for a minimum of 90 minutes. Disinfectants for Use in the Waste Tank The following disinfectant types may be used in the waste tank. It is pertinent that the appropriate type and quantity of disinfectant is placed in the waste tank to ensure effective inactivation of the biologics in use when the tank is full. Please check compatibility of combined products before use. 1. Sodium hypochlorite or bleach 2. Quaternary ammoniums 3. Thymols 4. Phenols CyAn ADP with Summit Software User Guide 115

116 CyAn ADP with Summit Software User Guide

Appendix B CyAn ADP Consumables and Parts Table B.1:Calibration Particles Code Product K0110 K0111 K0112 K0117 S2322 S2539 S2540 FluoroSpheres - 6-peak, 3.2 µm, 1 x 10 7 /ml, 2 ml/vial SpectrAlign - 3.0 µm, 1 x 10 7 /ml, 2 ml/vial FluoroSpheres - 8-peak, 3.0 µm, 1 x 10 7 /ml, 5 ml/vial SpectraComp Blank Particles, 5 ml/vial Sheath Fluid for Flow Cytometry, 20 Liter Water, ASTM Type 1-A, PuraFlo, 20 Liter Sheath Fluid 1X, PuraFlo, 20 Liter Table B.2: Decontamination/Clean/Rinse Solutions Code Product S2323 S2324 Solution, Clean & Rinse 5 Liter Solution, Decontamination 5 Liter CyAn ADP with Summit Software User Guide 117

Table B.3: CyAn ADP Filter Assemblies Code Product 10-40016 720/40 Filter Assy, PE-Cy5.5 10-40019 530/40 Filter Assy, FITC 10-40020 488/10 Filter Assy, SSC 10-40021 450/50 Filter Assy, HO 10-40022 400/40 Filter Assy, INDO-1 10-40029 488/UV Mirror Assy, Pos 6 10-40030 95/5BS/UV/Red Mirror Assy, Pos 1 10-40033 665/20 Filter Assy, APC 10-40034 613/20 Filter Assy, PE-TxRed 10-40035 575/25 Filter Assy, PE 10-40036 515/30 Filter Assy, FITC 10-40037 750LP Filter Assy, PE-Cy7 10-40038 680/30 Filter Assy PE-Cy5 10-40039 595DLP/730DLP Filter Assy, Pos 3 10-40040 545DLP/650DLP Filter Assy, Pos 2 10-40041 730DLP/425DLP Filter Assy, Pos 5 10-40042 640DLP/Mirror Filter/Mirror Assy, Pos 4 10-40053 665/20 Filter Assy, PE-Cy5 10-40054 750LP Filter Assy, APC-Cy7 10-40055 530/40 Filter Assy, CASY 10-40056 730DLP/485DLP Filter Assy, Pos 5 10-40057 700/20 Filter Assy, PE-Cy5.5 To order CyAn ADP consumables and parts, contact Dako Sales at 1.800.822.9902. 118 CyAn ADP with Summit Software User Guide

Appendix C Technical & Instrument Specifications The technical and instrument specifications for the CyAn ADP are summarized in the following table. Table C.1: CyAn ADP Data Sheet Performance Maximum processing speed Up to 70,000 events/sec Excitation Optics Optical parameters Beam geometry 2 scatter and 9 fluorescence Elliptical Number of excitation lines 3 Laser options nominal operating output (See Coherent OPSL Operator s Manual for more information.) Laser maximum output value (Accessible in the interior of instrument) Warm-up time 488 nm (20 mw Semiconductor), 635 nm (25 mw semiconductor), 405 nm (25 mw semiconductor) 488nm - 20mW 635nm 27.5mW 405 nm 27.5mW 30 minutes Detectors and Filters (User-selected) 488 nm Excitation FL1-530/40 nm, FL2-575/25 nm, FL3-613/20 nm, FL4-680/30 nm, FL5-750 nm 405 nm Excitation FL6-450/50 nm, FL7-530/40 635 nm Excitation FL8-665/20 nm, FL9-750 LP CyAn ADP with Summit Software User Guide 119

Signal Processing Compensation 9 x 9 full matrix with auto compensation Signal resolution 65536 (Summit software displays up to 4096 channels on all parameters) Data acquisition channels 11 Sensitivity MESF FITC < 100, MESF PE < 50 Fluidics Sheath Management System Sample flow rate Sheath fluid Quartz cuvette Noise level Controls sheath, waste, and cleaning fluids. Software, hardware, and smart-sensor controls with RUN/PAUSE modes. Stable control of flow rates from 0 to 5 µm/second. Up to 150 µl/min (2.5µL/sec) Maximum 1.03 bar (15 psi), nominal 0.41 bar (6 psi) UV-grade fused silica with 250 µm squaresectioned internal channel < 70 db Summit Workstation Platform Microsoft Windows XP Processor > 2 GHz, AMD Athlon, Intel Pentium Memory 1 GB RAM (minimum) Storage space 160 GB hard drive (minimum), 16X DVD +/- CDRW Monitor Network 24-inch LCD flat screen High-speed Ethernet 10/100 Mbps 120 CyAn ADP with Summit Software User Guide

Table C.2: CyAn ADP Instrument Specifications CyAn ADP Enclosure Type Installation Molded RIM polyurethane structural foam Indoor only Height 39.1cm (15.4 in) front cover closed 72.1cm (28.4 in) front cover open Dimensions (not including Utility Cart or Summit Workstation) Width - 33.3cm (13.1 in) Depth 49.8cm (19.6 in) 62.5cm (24.6 in) including clearance for cables. Weight - 36.3 kg (80 lbs) Auxiliary Components Sheath Management System (with casters): Houses sheath container, waste container, cleaner fluid container, air compressor, laser power supply, and vacuum Uninterruptible Power Supply (Optional) Summit Workstation External Transformer Height 61.3 cm (24.2 in) Width - 73.3 cm (28.9 in) Depth 61.9 cm (24.4 in) Weight 52 kg (115 lbs) Height 20.3 cm (7.9 in) Width 14.7 cm (5.7 in) Depth 44.5 cm (17.5 in) Weight 20 kg (44 lbs) Height - 42.9cm (16.9 in) Width 19.1 cm (7.5 in) Depth - 45.7cm (18.0 in) Weight - 10.5 kg (23 lbs) Height 19.1cm (7.5 in) Width 31.2 cm (12.3 in) Depth - 19.1 cm (7.5 in) Weight - 11.4 kg (25 lbs) CyAn ADP with Summit Software User Guide 121

Safety Interlock The front cover is protected with dual magnetic positive-break ( ) type interlock switch. Operates dual spring solenoid shutter actuators on all laser paths. Laser product class CLASS 1 LASER PRODUCT IEC/EN 60825-1/A2:2001 Laser light leakage Conforms to 21CFR1040.10 and 21CFR1040.11 Operating Environment Ambient temperature Relative humidity 15 to 30 C (59 to 86 F) For optimum performance maintain at +/- 2 C 20 to 80% RH (non-condensing) CyAn ADP System Utility Requirements & Fusing Power, standard ADP 115 VAC +/- 10%, 60 Hz, single phase, 1.75A Fuse Power, Sheath Management System Fuse, Sheath Management System Power, Summit Workstation (not fused) Power, Summit Workstation Monitor (not fused) Power, External Transformer Fuse, External Transformer power Type 5x20mm, low breaking, high capacity, IEC. 115 VAC operation 6.3A 100-230 VAC, 50/60 Hz, single phase, 2A Type 5x20mm, low breaking, high capacity, UL. 115 VAC operation 3A 115 VAC +/- 10%, 60 Hz, single phase, 1A 115 VAC +/- 10%, 60 Hz, single phase, 2A Input: 230 VAC +/- 10%, 50/60 Hz, 5A Output: 115 VAC +/- 10%, 60 Hz, 8A Type 5x20mm, low breaking, high capacity, IEC, 115 VAC operation 6.3A 122 CyAn ADP with Summit Software User Guide

Appendix D Symbol Definitions Instrument Serial Number Dako Model Number Date of Manufacture Alternating Current Input Fuse Caution, Consult Accompanying Documents Identification of Authorized Representative in the European Community Identification of Manufacturer Correct Disposal of this Product (according to Directive 2002/96/EC on Waste Electrical and Electronic Equipment [WEEE] applicable in the European Union and other European countries with separate collection systems). Contact a Dako representative for disposal of the equipment at the end of its working life. This product should not be mixed with other commercial waste for disposal. CyAn ADP with Summit Software User Guide 123

124 CyAn ADP with Summit Software User Guide

Appendix E Flow Cytometry References Researchers around the world and across many scientific disciplines rely on the MoFlo High- Performance Cell Sorter and the CyAn ADP High-Performance Flow Cytometer. A partial list of peer-reviewed journal articles highlighting some of their cutting-edge science on the CyAn ADP and MoFlo instruments follows. Bacteria & other microorganisms Bacteria have broad scientific relevance in today s research laboratory. Whether they are of interest for traditional microbiology, as a system for high-throughput screening, or as a vehicle for gene expression studies, their unique characteristics make them well suited for study by flow cytometry. With superior resolution for small particles, the CyAn ADP High-Performance Analyzer and the MoFlo High-Performance Cell Sorter offer fast, versatile, high-resolution platforms for evaluating and manipulating bacteria, viruses, fungi, protists, and other microorganisms. CyAn ADP and MoFlo University of Georgia Martin, D.L. and R.L. Tarleton. 2005. Antigen-specific T cells maintain an effector memory phenotype during persistent Trypanosoma cruzi infection. J Immunol 174(3): 1594-1601. CyAn ADP Colorado State University Bosio, C.M., A. Goodyear, and S. Dow. 2005. Early interaction of Yersinia Pestis with APCs in the lung. J Immunol 175(10): 6750-6756. MoFlo Brigham and Women s Hospital Sierig, G., C. Cywes, M.R. Wessels, and C.D. Ashbaugh. 2003. Cytotoxic effects of Streptolysin O and Streptolysin S enhance the virulence of poorly encapsulated Group A Streptococci. Infect Immun 71: 446. Gryllos, I., C. Cywes, M.H. Shearer, M. Cary, R.C. Kennedy, and M.R. Wessels. 2001. Regulation of capsule gene expression by group A Streptococcus during pharyngeal colonization and invasive infection. Mol Microbiol 42(1): 61-74. Centre for Environmental Research, Leipzig-Halle Müller, S. and A. Lösche. 2004. Population profiles of a commercial yeast strain in the course of brewing. J Food Eng 63(4): 375-381. Achilles, J., S. Müller, T. Bley, and W. Babel. 2004. Affinity of single S. cerevisiae cells to 2-NBDglucose under changing substrate concentrations. Cytometry Part A 61A(1): 88-98. CyAn ADP with Summit Software User Guide 125

Georg-August-Universität Adams, T.M., A. Wentzel, and H. Kolmar. 2005. Intimin-mediated export of passenger proteins requires maintenance of a translocation-competent conformation. J Bacteriol 187(2): 522-533. Jedrusik, M.A. and E. Schulze. 2003. Telomeric position effect variegation in Saccharomyces cerevisiae by Caenorhabditis elegans linker histones suggests a mechanistic connection between germ line and telomeric silencing. Mol Cell Biol 23(10): 3681-3691. Wentzel, A., A. Christmann, T. Adams, and H. Kolmar. 2001. Display of passenger proteins on the surface of Escherichia coli K-12 by the enterohemorrhagic E. coli intimin EaeA. J Bacteriol 183: 7273. Christmann, A., K. Walter, A. Wentzel, R. Kratzner, and H. Kolmar. 1999. The cystine knot of a squash-type protease inhibitor as a structural scaffold for E. coli cell surface display of conformationally constrained peptides. Protein Eng 12(9): 797-806. Wentzel, A., A. Christmann, R. Kratzner, and H. Kolmar. 1999. Sequence requirements of the GPNG β-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening. J Biol Chem 274(30): 21037-21043. Max Planck Institute Behrens, S., B.M. Fuchs, F. Mueller, and R. Amann. 2003. Is the in situ accessibility of the 16S rrna of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit? Appl Envir Microbiol 69(8): 4935-4941. Stanford University Wehrman, T., B. Kleaveland, J.H. Her, R.F. Balint, and H.M. Blau. 2002. Protein-protein interactions monitored in mammalian cells via complementation of β-lactamase enzyme fragments. PNAS 99: 3469. University of Georgia Gubbels, M.-J., B. Striepen, N. Shastri, M. Turkoz, and E.A. Robey. 2005. Class I major histocompatibility complex presentation of antigens that escape from the parasitophorous vacuole of Toxoplasma gondii. Infect Immun 73(2): 703-711. Worden, A. Z., S.W. Chisholm, and B.J. Binder. 2000. In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. with rrna-targeted peptide nucleic acid probes. Appl Environ Microbiol 66: 284. University of Illinois, Urbana Starwalt, S.E., E.L. Masteller, J.A. Bluestone, and D.M. Kranz. 2003. Directed evolution of a single-chain class II MHC product by yeast display. Protein Eng 16(2): 147-156. Cancer Although cancer, an extremely complex disease process involving genetic, environmental and behavioral factors, can stem from a vast range of cell types, it displays the common characteristic of uncontrolled cell division resulting in abnormal tissue growth. By enabling researchers to obtain specific cell cycle, phenotypic, and functional information about the mechanism in which cells become malignant and the body s response, the CyAn ADP High-Performance Analyzer and the MoFlo High-Performance Cell Sorter ultimately contribute to the development of cutting-edge cancer treatments and novel vaccines. CyAn ADP ARNAS Civico-Benfratelli, Palermo Gervasi, F., R. Lo Verso, C. Giambanco, G. Cardenale, C. Tomaselli, G. Pagnucco.2004. Flow cytometric immunophenotyping analysis of patterns of antigen expression in non-hodgkin's B cell lymphoma in samples obtained from different anatomic sites. Ann NY Academy Sci 1028(1): 457-462. 126 CyAn ADP with Summit Software User Guide

Northwestern University Medical School Goolsby, C., M. Paniagua, M. Tallman, R.B. Gartenhaus. 2005. Bcl-2 regulatory pathway is functional in chronic lymphocytic leukemia. Cytometry Part B: Clin Cytometry. 63B(1): 36-46. Jacobberger, JW, R.M. Sramkoski, P.S. Frisa, P.P. Ye, M.A. Gottlieb, D.W. Hedley, T.V. Shankey, B.L. Smith, M. Paniagua, C.L. Goolsby. 2003. Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity. Cytometry. 54A(2): 75-88. University of Pennsylvania Suzuki, E., V. Kapoor, H. Cheung, L.E. Ling, P.A. DeLong, L.R. Kaiser, S.M. Albelda. 2004. Soluble type II transforming growth factor-beta receptor inhibits established murine malignant mesothelioma tumor growth by augmenting host antitumor immunity. Clin Cancer Res 10(17): 5907-5918. MoFlo Baylor College of Medicine Wulf, G.G., R.Y. Wang, I. Kuehnle, D. Weidner, F. Marini, M.K. Brenner, M. Andreeff, and M.A. Goodell. 2001. A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood 98(4): 1166-73. Cancer Research UK Robinson, S.C., K.A. Scott, J.L. Wilson, R.G. Thompson, A.E.I. Proudfoot, and F.R. Balkwill. 2003. A chemokine receptor antagonist inhibits experimental breast tumor growth. Cancer Res 63(23): 8360-8365. Commissariat à l'energie Atomique Baghdoyan, S., J. Lamartine, D. Castel, A. Pitaval, Y. Roupioz, N. Franco, M. Duarte, M.T. Martin, and X. Gidrol. 2005. Id2 reverts cell cycle arrest induced by gamma irradiation in human HaCat keratinocytes. J Biol Chem 280(16): 15836-15841. City of Hope National Medical Center Cooper, L.J.N., Z. Al-Kadhimi, L.M. Serrano, T. Pfeiffer, S. Olivares, A. Castro, W.-C. Chang, S. Gonzalez, D. Smith, S.J. Forman, and M.C. Jensen. 2005. Enhanced antilymphoma efficacy of CD19-redirected influenza MP1-specific CTLs by cotransfer of T cells modified to present influenza MP1. Blood 105(4): 1622-1631. Holtz, M. S., M.L. Slovak, F. Zhang, C.L. Sawyers, S.J. Forman, and R. Bhatia. 2002. Imatinib mesylate (STI571) inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormally increased proliferation. Blood 99(10): 3792-800. Harvard Medical School Vonderheide, R.H., S.M. Domchek, J.L. Schultze, D.J. George, K.M. Hoar, D.-Y. Chen, K.F. Stephans, K. Masutomi, M. Loda, Z. Xia, K.S. Anderson, W.C. Hahn, and L.M. Nadler. 2004. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes. Clin Cancer Res 10(3): 828-839. Xia, J., Y. Tanaka, S. Koido, C. Liu, P. Mukherjee, S.J. Gendler, and J. Gong. 2003. Prevention of spontaneous breast carcinoma by prophylactic vaccination with dendritic/tumor fusion cells J Immunol 170(4): 1980-1986. Ghia, P., P. Transidico, J.P. Veiga, C. Schaniel, F. Sallusto, K. Matsushima, S.E. Sallan, A.G. Rolink, A. Mantovani, L.M. Nadler, and A.A. Cardoso. 2001. Chemoattractants MDC and TARC are secreted by malignant B-cell precursors following CD40 ligation and support the migration of leukemia-specific T cells. Blood 98(3): 533-40. Institut Pasteur Viguier, M., F. Lemaitre, O. Verola, M.-S. Cho, G. Gorochov, L. Dubertret, H. Bachelez, P. Kourilsky, and L. Ferradini. 2004. Foxp3 expressing CD4+CD25-high regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells. J Immunol 173(2): 1444-1453. CyAn ADP with Summit Software User Guide 127

Max Planck Institute Flemming, A., T. Brummer, M. Reth, and H. Jumaa. 2003. The adaptor protein SLP-65 acts as a tumor suppressor that limits pre-b cell expansion. Nature Immunol 4: 38-43. Memorial Sloan-Kettering Cancer Center Muriglan, S.J., T. Ramirez-Montagut, O. Alpdogan, T.W. van Huystee, J.M. Eng, V.M. Hubbard, A.A. Kochman, K.H. Tjoe, C. Riccardi, P. Paolo Pandolfi, S. Sakaguchi, A.N. Houghton, and M.R.M. van den Brink. 2004. GITR activation induces an opposite effect on alloreactive CD4+ and CD8+ T cells in graft-versus-host disease. J Exp Med 200(2): 149-157. Rego, E.M., L.Z. He, R.P. Warrell, Z.-G. W., and P. Paolo Pandolfi. 2000. Retinoic acid (RA) and As2O3 treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature of the leukemogenic process induced by the PML-RARalpha and PLZF-RARalpha oncoproteins. PNAS 97(18): 10173-8. Ontario Cancer Institute Zhang, L. and R. P. Hill 2004. Hypoxia enhances metastatic efficiency by up-regulating Mdm2 in KHT cells and increasing resistance to apoptosis. Cancer Res 64(12): 4180-4189. Sanquin Research at CLB, Amsterdam Mackus, W.J.M., A.P. Kater, A. Grummels, L.M. Evers, B. Hooijbrink, M.H.H Kramer, J.E. Castro, T.J. Kipps, R.A.W. van Lier, M.H.J. van Oers, and E. Eldering. 2005. Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation. Leukemia. Technical University of Munich Fleischer, K., B. Schmidt, W. Kastenmuller, D.H. Busch, I. Drexler, G. Sutter, M. Heike, C. Peschel, H. Bernhard. 2004. Melanoma-reactive class I-restricted cytotoxic T cell clones are stimulated by dendritic cells loaded with synthetic peptides, but fail to respond to dendritic cells pulsed with melanoma-derived heat shock proteins in vitro. J Immunol 172(1): 162-169. University of Texas MD Anderson Cancer Center Kochenderfer, J.N., S. Kobayashi, E.D. Wieder, C. Su, and J.J. Molldrem. 2002. Loss of T-lymphocyte clonal dominance in patients with myelodysplastic syndrome responsive to immunosuppression. Blood 100(10): 3639-3645. Guo, W., A. Pui-Yee Chan, H. Liang, E.D. Wieder, J.J. Molldrem, L.D. Etkin, and L. Nagarajan. 2002. A human Mix-like homeobox gene MIXL shows functional similarity to Xenopus Mix.1. Blood 100(1): 89-95. Walter and Eliza Hall Institute Scott, C.L., M. Schuler, V.S. Marsden, A. Egle, M. Pellegrini, D. Nesic, S. Gerondakis, S.L. Nutt, D.R. Green, and A. Strasser. 2004. Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation. J Cell Biol 164(1): 89-96. Washington University School of Medicine Rettig, M.P., J.K. Ritchey, J.L. Prior, J.S. Haug, D. Piwnica-Worms, and J.F. DiPersio. 2004. Kinetics of in vivo elimination of suicide gene-expressing T cells affects engraftment, graft-versus-host disease, and graft-versus-leukemia after allogeneic bone marrow transplantation. J Immunol 173(6): 3620-3630. 128 CyAn ADP with Summit Software User Guide

Dendritic cells Dendritic cells the most potent antigen-presenting cells in the immune system typically comprise less than 2% of lymphoid organs. The CyAn ADP High-Performance Analyzer rapidly characterizes this complex population and the MoFlo High-Performance Cell Sorter quickly and accurately purifies these rare cells, providing a functional end product for use in further investigations. CyAn ADP and MoFlo Colorado State University Perry, J.A., A. Rush, R.J. Wilson, C.S. Olver, A.C. Avery. 2004. Dendritic cells from malaria-infected mice are fully functional APC. J Immunol 172(1): 475-482. University of California, San Francisco Lohr, J., B. Knoechel, E.C. Kahn, and A.K. Abbas. 2004. Role of B7 in T Cell Tolerance. J Immunol 173(8): 5028-5035. CyAn ADP Children s Hospital of Philadelphia Wang, L., R. Han, I. Lee, A. Hancock, G. Xiong, M. Gunn and W. Hancock. 2005. Permanent survival of fully MHC- Mismatched islet allografts by targeting a single chemokine receptor pathway. J. of Immunol 175(10): 6311-6318. Colorado State University Bisio, C. and S. Dow. 2005. Francisella tularensis induces aberrant activation of pulmonary dendritic cells. J. of Immunol 175(10): 6792-6801. Wistar Institute Hensley, S., W. Giles-Davis, K. McCoy, W. Weninger, and H. Ertl. 2005. Dendritic cell maturation, but not CD8 + T Cell induction, is dependent on type I IGN signaling during vaccination with adenovirus vectors. J. of Immunol 175(9): 6032-6041. MoFlo Amgen Brawand, P., D.R. Fitzpatrick, B.W. Greenfield, K. Brasel, C.R. Maliszewski, and T. De Smedt. 2002. Murine plasmacytoid pre-dendritic cells generated from Flt3 ligand-supplemented bone marrow cultures are immature APCs. J Immunol 169: 6711. Basel Institute for Immunology Schaniel, C.,L. Bruno, F. Melchers, and A.G. Rolink. 2002. Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5-deficient pre-b I-cell clones. Blood 99: 472. Barchet, W., M. Cella, B. Odermatt, C. Asselin-Paturel, M. Colonna, and U. Kalinke. 2002. Virus-induced interferon-alpha production by a dendritic cell subset in the absence of feedback signaling in vivo. J Exp Med 195: 507. CellTech Nakagawa, T.Y., W.H. Brissette, P.D. Lira, R.J. Griffiths, N. Petrushova, J. Stock, J.D. McNeish, S.E. Eastman, E.D. Howard, S.R.M. Clarke, E.F. Rosloniec, E.A. Elliott, and A.Y. Rudensky. 1999. Impaired invariant chain degradation and antigen presentation and diminished collagen-induced arthritis in Cathepsin S null mice. Immunity 10: 207-217. CyAn ADP with Summit Software User Guide 129

Dana-Farber Cancer Institute Shigematsu, H., B. Reizis, H. Iwasaki, S. Mizuno, D. Hu, D. Traver, P. Leder, N. Sakaguchi, and K. Akashi. 2004. Plasmacytoid dendritic cells activate lymphoid-specific genetic programs irrespective of their cellular origin. Immunity 21: 43-53. Briken, V., R.M. Jackman, G.F.M. Watts, R.A. Rogers, and S.A. Porcelli. 2000. Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens. J Exp Med 192: 281. Edward Jenner Institute Montoya, M., M. J. Edwards, D.M. Reid, and P. Borrow. 2005. Rapid activation of spleen dendritic cell subsets following lymphocytic choriomeningitis virus infection of mice: analysis of the involvement of type l IFN 1. J Immunol 174(4): 1851-1861. Montoya, M., G. Schiavoni, F. Mattei, I. Gresser, F. Belardelli, P. Borrow, and D.F. Tough. 2002. Type l interferons produced by dendritic cells promote their phenotypic and functional activation. Blood 99: 3263. Le Bon, A., G. Schiavoni, G. D'Agostino, I. Gresser, F. Belardelli, and D.F. Tough. 2001. Type l interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity 14: 461. GKT Medical School, Guy s Campus Wang, Y., C.G. Kelly, J.T. Karttunen, T. Whittall, P.J. Lehner, L. Duncan, P. MacAry, J.S. Younson, M. Singh, W. Oehlmann, G. Cheng, L. Bergmeier, and T. Lehner. 2001. CD40 is a cellular receptor mediating mycobacterial heat shock protein 70 stimulation of CC-chemokines. Immunity 15: 971. Harvard Medical School Turley, S., L. Poirot, M. Hattori, C. Benoist, and D. Mathis. 2003. Physiological {beta} cell death triggers priming of selfreactive T cells by dendritic cells in a type-1 diabetes model. J Exp Med 198(10): 1527-1537. Imperial Cancer Research Fund Edwards, A. D., D. Chaussabel, S. Tomlinson, O. Schulz, A. Sher, and C. Reis e Sousa, Caetanol. 2003. Relationships among murine CD11c-high dendritic cell subsets as revealed by baseline gene expression patterns. J Immunol 171(1): 47-60. Edwards, A.D., S.P. Manickasingham, R. Sporri, S.S. Diebold, O. Schulz, A. Sher, T. Kaisho, S. Akira, and C. Reis e Sousa. 2002. Microbial recognition via toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering. J Immunol 169: 3652. Reis e Sousa, C., G. Yap, O. Schulz, N. Rogers, M. Schito, J. Aliberti, S. Hieny, and A. Sher. 1999. Paralysis of dendritic cell IL-12 production by microbial products prevents infection-induced immunopathology. Immunity 11: 637. Institut Pasteur Moron, G., P. Rueda, I. Casal, and C. Leclerc. 2002. CD8alpha- CD11b+ dendritic cells present exogenous virus-like particles to CD8+ T cells and subsequently express CD8alpha and CD205 molecules. J Exp Med 195: 1233. Ludwig Institute for Cancer Research, Victoria Schnurr, M., T. Toy, A. Shin, M. Wagner, J. Cebon, and E. Maraskovsky. 2005. Extracellular nucleotide signaling by P2 receptors inhibits IL-12 and enhances IL-23 expression in human dendritic cells: a novel role for the camp pathway. Blood 105(4): 1582-1589. Jefford, M., M. Schnurr, T. Toy, K. Masterman, A. Shin, T. Beecroft, T.Y. Tai, K. Shortman, M. Shackleton, I.D. Davis, P. Parente, T. Luft, W. Chen, J. Cebon, and E. Maraskovsky. 2003. Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli. Blood 102(5): 1753-1763. Schnurr, M., T. Toy, P. Stoitzner, P. Cameron, A. Shin, T. Beecroft, I.D. Davis, J. Cebon, and E. Maraskovsky. 2003. ATP gradients inhibit the migratory capacity of specific human dendritic cell types: Implications for P2Y 11 receptor signaling. Blood 102(2): 613-620. 130 CyAn ADP with Summit Software User Guide

Memorial Sloan-Kettering Cancer Center Pillarisetty, V.G., A.B. Shah, G. Miller, J.I. Bleier, and R.P. DeMatteo. 2004. Liver dendritic cells are less immunogenic than spleen dendritic cells because of differences in subtype composition. J Immunol 172(2): 1009-1017. Mt. Sinai School of Medicine Qu, C., T.M. Moran, and G.J. Randolph. 2003. Autocrine type I IFN and contact with endothelium promote the presentation of influenza A virus by monocyte-derived APC. J Immunol 170: 1010. National Institute for Medical Research Boonstra, A., C. Asselin-Paturel, M. Gilliet, C. Crain, G. Trinchieri, Y.J. Liu, and A. O Garra. 2003. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper Type 1 and 2 cell development: Dependency on antigen dose and differential toll-like receptor ligation. J Exp Med 197(1): 101-109. Oxford University Salio, M., M.J. Palmowski, A. Atzberger, I.F. Hermans, and V. Cerundolo. 2004. CpG-matured murine plasmacytoid dendritic cells are capable of in vivo priming of functional CD8 T cell responses to endogenous but not exogenous antigens. J Exp Med 199(4): 567-579. Stanford University School of Medicine Karsunky, H., M. Merad, A. Cozzio, I.L. Weissman, and M.G. Manz. 2003. Flt3 ligand regulates dendritic cell development from Flt3+ lymphoid and myeloid-committed progenitors to Flt3+ dendritic cells in vivo. J Exp Med 198(2): 305-313. Technical University of Munich Heit, A., F. Schmitz, M. O Keeffe, C. Staib, D.H. Busch, H. Wagner, and K.M. Huster. 2005. Protective CD8 T cell immunity triggered by CpG-protein conjugates competes with the efficacy of live vaccines. J Immunol 174(7): 4373-4380. Hochrein, H., B. Schlatter, M. O'Keeffe, C. Wagner, F. Schmitz, M. Schiemann, S. Bauer, M. Suter, and H. Wagner. 2004. Herpes simplex virus type-1 induces IFN-{alpha} production via Toll-like receptor 9-dependent and -independent pathways. PNAS 101(31): 11416-11421. Spies, B., H. Hochrein, M. Vabulas, K. Huster, D.H. Busch, F. Schmitz, A. Heit and H. Wagner. 2003. Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice. J Immunol 171(11): 5908-5912. Alferink, J., I. Lieberam, W. Reindl, A. Behrens, S. Weiss, N. Hüser, K. Gerauer, R. Ross, A.B. Reske-Kunz, P. Ahmad- Nejad, H. Wagner, and I. Förster. 2003. Compartmentalized production of CCL17 in vivo: Strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen. J Exp Med 197(5): 585-599. University of California, San Francisco Fong, L., M. Mengozzi, N.W. Abbey, B.G. Herndier, and E.G. Engleman. 2002. Productive infection of plasmacytoid dendritic cells with human immunodeficiency virus type 1 is triggered by CD40 ligation. J Virol 76: 11033. University of Chicago Chun, T. M.J. Page, L. Gapin, J.L. Matsuda, H. Xu, H. Nguyen, H.S. Kang, A.K. Stanic, S. Joyce, W.A. Koltun, M.J. Chorney, M. Kronenberg, and C.R. Wang. 2003. CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells. J Exp Med 197(7): 907-918. University of North Carolina, Chapel Hill Yang. O.O., F.K. Racke, P.T. Nguyen, R. Gauslking, M.E. Severino, H.F. Horton, M.C. Byrne, J.L. Stroninger, and S. B. Wilson. 2000. CD1d on myeloid dendritic cells stimulates cytokine secretion from and cytolytic activity of Vα24JαQ T cells: A feedback mechanism for immune regulation. J Immunol 165: 3756. CyAn ADP with Summit Software User Guide 131

Walter and Eliza Hall Institute Naik, S.H., A.I. Proietto, N.S. Wilson, A. Dakic, P. Schnorrer, M. Fuchsberger, M.H. Lahoud, M. O Keeffe, Q. Shao, W. Chen, J.A. Villadangos, K. Shortman, and L. Wu. 2005. Cutting edge: Generation of splenic CD8+ and CD8- dendritic cell equivalents in Fms-like tyrosine kinase 3 ligand bone marrow cultures. J Immunol 174(11): 6592-6597. Baldwin, T., S. Henri, J. Curtis, M. O'Keeffe, D. Vremec, K. Shortman, and E. Handman. 2004. Dendritic cell populations in Leishmania major-infected skin and draining lymph nodes. Infect Immun 72(4): 1991-2001. Dakic, A., Q. Shao, A. D Amico, M. O Keeffe, W. Chen, K. Shortman, and L. Wu. 2004. Development of the dendritic cell system during mouse ontogeny. J Immunol 172(2): 1018-1027. O'Keeffe, M., H. Hochrein, D. Vremec, B. Scott, P. Hertzog, L. Tatarczuch, and K. Shortman. 2003. Dendritic cell precursor populations of mouse blood: identification of the murine homologues of human blood plasmacytoid pre-dc2 and CD11c+ DC1 precursors. Blood 101(4): 1453-1459. Henri, S., J. Curtis, H. Hochrein, D. Vremec, K. Shortman, and E. Handman. 2002. Hierarchy of susceptibility of dendritic cell subsets to infection by Leishmania major: Inverse relationship to interleukin-12 production. Infect Immun 70: 3874. O'Keeffe, M., H. Hochrein, D. Vremec, I. Caminschi, J.L. Miller, E.M. Anders, L. Wu, M.H. Lahoud, S. Henri, B. Scott, P. Hertzog, L. Tatarczuch, and K. Shortman. 2002. Mouse plasmacytoid cells: Long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8+ dendritic cells only after microbial stimulus. J Exp Med 196: 1307. O Keeffe, M., H. Hochrein, D. Vremec, J. Pooley, R. Evans, S. Woulfe, and K. Shortman. 2002. Effects of administration of progenipoietin 1, Flt-3 ligand, granulocyte colony-stimulating factor, and pegylated granulocyte-macrophage colonystimulating factor on dendritic cell subsets in mice. Blood 99(6): 2122-2130. O'Keeffe, M., H. Hochrein, D. Vremec, B. Scott, P. Hertzog, L. Tatarczuch, and K. Shortman. 2002. Dendritic cell precursor populations of mouse blood: Identification of the murine homologues of human blood plasmacytoid pre-dc2 and CD11c+ DC1 precursors. Blood 101(4): 1453-1459. Riese, R. J., G.P. Shi, J. Villadangos, D. Stetson, C. Driessen, A. M. Lennon-Dumenil, C. L. Chu, Y. Naumov, S. M. Behar, H. Ploegh, R. Locksley, and H. A. Chapman. 2001. Regulation of CD1 function and NK1.1(+) T cell selection and maturation by cathepsin S. Immunity 15: 909. Vandenabeele, S., H. Hochrein, N. Mavaddat, K. Winkel, and K. Shortman. 2001. Human thymus contains 2 distinct dendritic cell populations. Blood 97: 1733. Villadangos, J. A., M. Cardoso, R. J. Steptoe, D. van Berkel, J. Pooley, F.R. Carbone, and K. Shortman. 2001. MHC class II expression is regulated in dendritic cells independently of invariant chain degradation. Immunity 14: 739. Vremec, D., J. Pooley, H. Hochrein, L. Wu, and K. Shortman. 2000. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol 164: 2978-2986. Kamath, A.T., J. Pooley, M.A. O Keeffe, D. Vremec, Y. Zhan, A.M. Lew, A. D Amico, L. Wu, D.F. Tough, and K. Shortman. 2000. The development, maturation and turnover rate of mouse spleen dendritic cell populations. J Immunol 165: 6762-6770. Wu, L., A. D'Amico, K. D. Winkel, M. Suter, D. Lo, and K. Shortman. 1998. RelB is essential for the development of myeloid-related CD8alpha- dendritic cells but not of lymphoid-related CD8alpha+ dendritic cells. Immunity 9: 839. Drosophila cells Drosophila continues to be the organism of choice for many at the forefront of genomics and proteomics research. Evolutionary conservation and ease of genetic manipulation have made this organism an attractive model for the study of eukaryotic development mechanisms. With their superior resolution and multicolor capability, the CyAn ADP High-Performance Analyzer and the MoFlo High-Performance Cell Sorter are uniquely suited as tools for the study of complex biological processes. MoFlo European Molecular Biology Laboratory Hipfner, D.R., K. Weigmann, and S.M. Cohen. 2002. The bantam gene regulates Drosophila growth. Genetics 161: 1527. 132 CyAn ADP with Summit Software User Guide

Jasper, H., V. Benes, A. Atzberger, S. Sauer, W. Ansorge, and D. Bohmann. 2002. A genomic switch at the transition from cell proliferation to terminal differentiation in the Drosophila eye. Dev Cell 3: 511-521. Massachusetts General Hospital Harvey, K.F., C. M. Pfleger, and I.K. Hariharan, http://www.cell.com/content/article/abstract?uid=piis0092867403005579&highlight=moflo&highlight=drosophila - cor1#cor1. 2003. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell 114(4): 457-467. Tseng, A.S.K., and I.K. Hariharan. 2002. An overexpression screen in Drosophila for genes that restrict growth or cellcycle progression in the developing eye. Genetics 162:229. Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell 105:345. Bryant, Z., L. Subrahmanyan, M. Tworoger, L. LaTray, C.R. Liu, M.J. Li, G. van den Engh, and H. Ruohola-Baker. 1999. Characterization of differentially expressed genes in purified Drosophila follicle cells: Toward a general strategy for cell type-specific developmental analysis. PNAS 96:5559. Massachusetts Institute of Technology Neves, G., J. Zucker, M. Daly, and A. Chess. 2004. Stochastic yet biased expression of multiple Dscam splice variants by individual cells. Nature Genetics 36: 240-246. Stanford University School of Medicine Tirouvanziam, R., C.J. Davidson, J.S. Lipsick, and L.A. Herzenberg. 2004. Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes. PNAS 101(9): 2912-2917. University of California, Irvine Apostol, B.L., A. Kazantsev, S. Raffioni, K. Illes, J. Pallos, L. Bodai, N. Slepko, J.E. Bear, F.B. Gertler, S. Hersch, D.E. Housman, J.L. Marsh, and L.M. Thompson. 2003. A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila. PNAS 100(10) 5950-5955. University of North Carolina, Chapel Hill Cayirlioglu, P., W.O. Ward, S.C. Silver Key, and R.J. Duronio. 2003. Transcriptional repressor functions of Drosophila E2F1 and E2F2 cooperate to inhibit genomic DNA synthesis in ovarian follicle cells. Mol Cell Biol 23(6): 2123-2134. Noureddine, M.A., T.D. Donaldson, S.A. Thacker, and R.J. Duronio. 2002. Drosophila Roc1a encodes a RING-H2 protein with a unique function in processing the Hh signal transducer Ci by the SCF E3 ubiquitin ligase. Dev Cell 2:757. CyAn ADP with Summit Software User Guide 133

Washington University School of Medicine Read, R.D., E.A. Bach, and R.L. Cagan. 2004. Drosophila C-Terminal Src Kinase Negatively Regulates Organ Growth and Cell Proliferation through Inhibition of the Src, Jun N-Terminal Kinase, and STAT Pathways. Mol Cell Biol 24(15): 6676-6689. Fluorescent proteins In the rapidly evolving proteomics era, applications for fluorescent proteins, such as CFP, GFP, YFP and BFP, continue to grow. Whether as simple indicators of gene expression levels or as tools in fluorescence resonance energy transfer (FRET), the CyAn ADP High-Performance Analyzer and the MoFlo High-Performance Cell Sorter are ideal platforms for detecting these proteins and isolating cells with desired expression patterns. CyAn ADP Trudeau Institute Mayer, K. D., K. Mohrs, S.R. Crowe, L.L. Johnson, P. Rhyne, D.L. Woodland, and M. Mohrs. 2005. The functional heterogeneity of type 1 effector T cells in response to infection is related to the potential for IFN-gamma production. J Immunol 174(12): 7732-7739. MoFlo Aaron Diamond AIDS Research Center Roncarati, R. N. Sestan, M.H. Scheinfeld, B.E. Berechid, P.A. Lopez, O. Meucci, J.C. McGlade, P. Rakic, and L. D Adamio. 2002. The γ-secretase-generated intracellular domain of β-amyloid precursor protein binds Numb and inhibits Notch signaling. PNAS 99(10): 7102-7107. Scheinfeld, M.H., R. Roncarati, P. Vito, P.A. Lopez, M. Abdallah, and L. D Adamio. 2002. Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer s β-amyloid precursor protein (APP). J Biol Chem 277(5): 3767-3775. Albert Einstein College of Medicine Stadtfeld, M. and T. Graf. 2005. Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing. Devel 132: 203-213. Children s Hospital, Boston Gartner, F., F.W. Alt, R.J. Monroe, and K.J. Seidl. 2000. Antigen-independent appearance of recombination activating gene (RAG)-positive bone marrow B cells in the spleens of immunized mice. J Exp Med 192(12): 1745-1754. Monroe, R. J., K.J. Seidl, F. Gaertner, S. Han, F. Chen, J. Sekiguchi, J. Wang, R. Ferrini, L. Davidson, G. Kelsoe, and F.W. Alt. 1999. RAG2:GFP knockin mice reveal novel aspects of RAG2 expression in primary and peripheral lymphoid tissues. Immunity 11: 201-212. Dana-Farber Cancer Institute Heck, S., O. Ermakova, H. Iwasaki, K. Akashi, C.W. Sun, T.M. Ryan, T. Townes, and T. Graf. 2003. Distinguishable live erythroid and myeloid cells in β-globin ECFP x lysozyme EGFP mice. Blood 101(3): 903-906. Rosen, E.D., C. Husi, X. Wang, S. Sakai, M.W. Freeman, F.J. Gonzalez, and B.M. Spiegelman. 2002. C/EBPα induces adipogenesis through PPARgamma: a unified pathway. Genes & Devel 16: 22-26. Mao, X., Y. Fujiwara, A. Chapdelaine, H. Yang, and S.H. Orkin. 2001. Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain. Blood 97(1): 324. Tomasson, M.H., I.R. Williams, S. Li, J. Kutok, D. Cain, S. Gillessen, G. Dranoff, R.A. Van Etten, and D.G. Gilliland. 2001. Induction of myeloproliferative disease in mice by tyrosine kinase fusion oncogenes does not require granulocytemacrophage colony-stimulating factor or interleukin-3. Blood 97(5): 1435. 134 CyAn ADP with Summit Software User Guide

Dorris, D.R., and K. Struhl. 2000. Artificial recruitment of TFIID, but not RNA polymerase II holoenzyme, activates transcription in mammalian cells. Mol Cell Biol 20(12): 4350-4358. John Hopkins University Tang, Q., and M. Edidin. 2003. Lowering the barriers to random walks on the cell surface. Biophysical J 84: 400-407. Massachusetts General Hospital Kishimoto, J., R.E. Burgeson, and B.A. Morgan. 2000. Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes & Devel 14(10): 1181-1185. Kishimoto, J., R. Ehama, L. Wu, S. Jiang, N. Jiang, and R.E. Burgeson. 1999. Selective activation of the versican promoter by epithelial-mesenchymal interactions during hair follicle development. PNAS 96: 7336-7341. Massachusetts Institute of Technology Hendricks, C. A., K. H. Almeida, M. S. Stitt, V. S. Jonnalagadda, R.E. Rugo, G.F. Kerrison, and B.P. Engelward. 2003. Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cdna direct repeat in transgenic mice. PNAS 100(11): 6325-6330. Medical Research Council Rada, C., J.M. Jarvis, and C. Milstein. 2002. AID-GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization. PNAS 99(10): 7003-7008. Mt. Sinai School of Medicine Uttamsingh, S., C.S. Zong, and L.H. Wang. 2003. Matrix-independent activation of PI3 kinase, State3 and Cyclin A- associated Cdk2 is essential for anchorage-independent growth of v-ros-transformed chicken embryo fibroblasts. J Biol Chem 278(21): 18798-18810. Olaso, E., K. Ikeda, F.J. Eng, L. Xu, L. Wang, H.C. Lin, and S.L. Friedman. 2001. DDR2 receptor promotes MMP-2- mediated proliferation and invasion by hepatic stellate cells. J Clin Investigation 108(9): 1369-1378. National Institutes of Health He, L., X. Wu, J. Simone, D. Hewgill, and P.E. Lipsky. 2005. Determination of tumor necrosis factor receptor-associated factor trimerization in living cells by CFP->YFP->mRFP FRET detected by flow cytometry. Nuc Acid Res 33(6): e61. He, L., X. Wu, F. Meylan, D.P. Olson, J. Simone, D. Hewgill, R. Siegel, and P.E. Lipsky. 2004. Monitoring caspase activity in living cells using fluorescent proteins and flow cytometry. Am J Pathol 164(6): 1901-1913. National Jewish Medical and Research Center Albig, A.R. and W.P. Schiemann. 2005. Identification and characterization of regulator of G protein signaling 4 (RGS4) as a novel Inhibitor of tubulogenesis: RGS4 inhibits mitogen-activated protein kinases and vascular endothelial growth factor signaling. Mol Biol Cell 16(2): 609-625. Oklahoma Medical Research Foundation Nakamura, K., A. Malykhin, and K.M. Coggeshall. 2002. The Src homology 2 domain-containing inositol 5-phosphatase negatively regulates Fcγ receptor-mediated phagocytosis through immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors. Blood 100(9): 3374-3382. Ontario Cancer Institute Badour, K., J. Zhang, F. Shi, Y. Leng, M. Collins, and K.A. Siminovitch. 2004. Fyn and PTP-PEST-mediated regulation of Wiskott-Aldrich Syndrome protein (WASp) tyrosine phosphorylation is required for coupling T cell antigen receptor engagement to WASp effector function and T cell activation. J Exp Med 199(1): 99-112. McGavin, M.K.H., K. Badour, L.A. Hardy, T.J. Kubiseksi, J. Zhang, and K.A. Siminovitch. 2001. The intersectin 2 adaptor links Wiskott Aldrich Syndrome protein (WASp)-mediated actin polymerization to T cell antigen receptor endocytosis. J Exp Med 194(12): 1777-1787. CyAn ADP with Summit Software User Guide 135

Regeneron Pharmaceuticals Byrne, A. T., L. Ross, J. Holash, M. Nakanishi, L. Hu, J.I. Hofmann, G.D. Yancopoulos, and R.B. Jaffe. 2003. Vascular endothelial growth factor-trap decreases tumor burden, inhibits ascites, and causes dramatic vascular remodeling in an ovarian cancer model. Clin Cancer Res 9(15): 5721-5728. Rommel, C., B.A. Clarke, S. Zimmermann, L. Nunez, R. Rossman, K. Reid, K. Moelling, G.D. Yancopoulos, and D.J. Glass. 1999. Differentiation stage-specific inhibition of the Raf-MIK-ERK pathway by Akt. Science 286: 1738-1741. Rigel Pharmaceuticals Chu, P. C., J. Wu, X.C. Liao, J. Pardo, H. Zhao, C. Li, M.K. Mendenhall, E. Pali, M. Shen, S. Yu, V.C. Taylor, G. Aversa, S. Molineaux, D.G. Payan, and E.S. Masuda. 2004. A novel role for p21-activated protein kinase 2 in T cell activation. J Immunol 172(12): 7324-7334. Kinsella, T. M., C. T. Ohashi, A. G. Harder, G. C. Yam, W. Li, B. Peelle, E. S. Pali, M. K. Bennett, S. M. Molineaux, D. A. Anderson, E. S. Masuda, and D. G. Payan. 2002. Retrovirally delivered random cyclic peptide libraries yield inhibitors of interleukin-4 signaling in human B cells. J Biol Chem 277:37512. Lorens, J.B., M.K. Bennett, D.M. Pearsall, W.R. Throndset, A.B. Rossi, R.J. Armstrong, B.P. Fox, E.H. Chan, Y. Luo, E. Masuda, D.A. Ferrick, D.C. Anderson, D.G. Payan, and G.P. Nolan. 2000. Retroviral delivery of peptide modulators of cellular functions. Mol Therapy 1(5): 438-447. Demo, S.D., E. Masuda, A.B. Rossi, B.T. Throndset, A.L. Gerard, E.H. Chan, R.J. Armstrong, B.P. Fox, J.b. Lorens, D.G. Pana, R.H. Scheller, and J.M. Fisher. 1999. Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-v binding assay. Cytometry 36: 340-348. St. Jude Children s Research Hospital Eischen, C.J., M.F. Roussel, S.J. Korsmeyer, and J.L. Cleveland. 2001. Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis. Mol Cell Biol 21(22): 7653-7662. University of California, San Francisco Voehringer, D., K. Shinkai, and R.M. Locksley. 2004. Type 2 immunity reflects orchestrated recruitment of cells committed to IL-4 production. Immunity 20: 267-277. Mohrs, M., K. Shinkai, K. Mohrs, and R.M. Locksley. 2001. Analysis of type 2 immunity in vivo with bicistronic IL-4 reporter. Immunity 15(2): 303-311. University of Chicago Allenspach, E.J., P. Cullinan, J. Tong, Q. Tang, A.G. Tesciuba, J.L. Cannon, S.M. Takahashi, R. Morgan, J.K. Burkhardt, and A.I. Sperling. 2001. ERM-dependent movement of CD43 defines a novel protein complex distal to the immunological synapse. Immunity 15: 739-750. University of North Carolina, Chapel Hill Alekseev, O.M., D.C. Bencic, R.T. Richardson, E.E. Widgren, and M.G. O Rand. 2003. Overexpression of the linker histone-binding protein tnasp affects progression through the cell cycle. J Biol Chem 278(10): 8846-8852. Buslepp, J., S.E. Kerry, D. Loftus, J.A. Frelinger, E. Appella, and E.J. Collins. 2003. High affinity xenoreactive TCR:MHC interaction recruits CD8 in absence of binding to MHC. J Immunol 170: 373-383. Washington University School of Medicine Hanson, P., V. Mathews, S.H. Marrus, and T.A. Graubert,. 2003. Enhanced green fluorescent protein targeted to the Sca- 1 (Ly-6A) locus in transgenic mice results in efficient marking of hematopoietic stem cells in vivo. Exp Hematol 31(2): 159-167. 136 CyAn ADP with Summit Software User Guide

High-throughput screening Interest is growing in the use of flow cytometers to screen cell- or bead-based combinatorial libraries. 1-3 Increasingly, flow cytometric assays are used to detect molecules that bind to a target protein in vitro or exhibit a particular activity in a cell-based assay. Flow cytometry also enables screening of protein libraries expressed in cells or displayed on the surface of bacteria or beads. A flow cytometer, for instance, can detect modulation of a signal transduction pathway by a particular small molecule and identify proteins with a particular binding specificity, enzymatic activity, expression level and stability. The CyAn ADP High-Performance Analyzer and the MoFlo High-Performance Cell Sorter are finding wide application in this arena. CyAn ADP Trudeau Institute Mayer, K. D., K. Mohrs, et al. 2005. The functional heterogeneity of type 1 effector T cells in response to infection is related to the potential for IFN-gamma production. J Immunol 174(12): 7732-7739. MoFlo Diversa Zengler, K., G. Toledo, M. Rappé, J. Elkins, E.J. Mathur, J.M. Short, and M. Keller. 2002. Cultivating the uncultured. PNAS 99(24): 15681-15686. Georg-August-Universität Wentzel, A., A. Christmann, T. Adams, and H. Kolmar. 2001. Display of passenger proteins on the surface of Escherichia coli K-12 by the enterohemorrhagic E. coli intimin EaeA. J Bacteriol 183: 7273. Christmann, A., K. Walter, A. Wentzel, R. Kratzner, and H. Kolmar. 1999. The cystine knot of a squash-type protease inhibitor as a structural scaffold for E. coli cell surface display of conformationally constrained peptides. Protein Eng 12(9): 797-806. Wentzel, A., A. Christmann, R. Kratzner, and H. Kolmar. 1999. Sequence requirements of the GPNG b-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening. J Biol Chem 274(30) 21037-21043. Lynx Therapeutics Brenner, S., M. Johnson, J. Bridgham, G. Golda, D.H. Lloyd, D. Johnson, S. Luo, S. McCurdy, M. Foy, M. Ewan, R. Roth, D. George, S. Elitr, G. Albrecht, E. Vermaas, S.R. Williams, K. Moon, T. Burcham, M. Pallas, R.B. DuBridge, J. Kirchner, K. Fearon, J. Mao, and K. Corcoran. 2000. Gene expression analysis by massively parallel signature sequencing on microbead arrays. Nature Biotechnol 18: 630-634. Brenner, S., S.R. Williams, E.H. Vermaas, T. Storck, K. Moon, C. McCollum, J. Mao, S. Luo, J.J. Kirchner, S. Eletr, R.B. DuBridge, T. Burcham, and G. Albrecht. 2000. In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cdnas. PNAS 97(4): 1665. Massachusetts Institute of Technology Luo, B., A. D. Heard, and H.F. Lodish. 2004. From the Cover: Small interfering RNA production by enzymatic engineering of DNA (SPEED). PNAS 101(15): 5494-5499. National Jewish Medical and Research Center Wang, Y., A. Rubtsov, R. Heiser, J. White, F. Crawford, P. Marrack, and J.W. Kappler. 2005. Using a baculovirus display library to identify MHC class I mimotopes. PNAS 102(7): 2476-2481. CyAn ADP with Summit Software User Guide 137

Rigel Pharmaceuticals Chu, P. C., J. Wu, X.C. Liao, J. Pardo, H. Zhao, C. Li, M.K. Mendenhall, E. Pali, M. Shen, S. Yu, V.C. Taylor, G. Aversa, S. Molineaux, D.G. Payan, and E.S. Masuda. 2004. A novel role for p21-activated protein kinase 2 in T cell activation. J Immunol 172(12): 7324-7334. Kinsella, T. M., C. T. Ohashi, A. G. Harder, G. C. Yam, W. Li, B. Peelle, E. S. Pali, M. K. Bennett, S. M. Molineaux, D. A. Anderson, E. S. Masuda, and D. G. Payan. 2002. Retrovirally delivered random cyclic peptide libraries yield inhibitors of interleukin-4 signaling in human B cells. J Biol Chem 277: 37512. Lorens, J.B., M.K. Bennett, D.M. Pearsall, W.R. Throndset, A.B. Rossi, R.J. Armstrong, B.P. Fox, E.H. Chan, Y. Luo, E. Masuda, D.A. Ferrick, D.C. Anderson, D.G. Payan, and G.P. Nolan. 2000. Retroviral delivery of peptide modulators of cellular functions. Mol Therapy 1(5): 438-447. Demo, S.D., E. Masuda, A..B. Rossi, B.T. Throndset, A.L. Gerard, E.H. Chan, R.J. Armstrong, B.P. Fox, J.B. Lorens, D.G. Payan, R.H. Scheller, and J.M. Fisher. 1999. Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-v binding assay. Cytometry 36: 340-348. University of Georgia Gubbels, M.-J., C. Li, and B. Striepen. 2003. High-throughput growth assay for Toxoplasma gondii using yellow fluorescent protein. Antimicrob Agents Chemother 47:309. University of North Carolina, Chapel Hill Chang, J. H., J. M. Urbach, T.F. Law, L.W. Arnold, A. Hu, S. Gombar, S.R. Grant, F.M. Ausubel, and J.L. Dangl. 2005. A high-throughput, near-saturating screen for type III effector genes from Pseudomonas syringae. PNAS: 102(7): 2549-2554. University of Queensland Lawrie, G.A., B.J. Battersby, and M. Trau. 2003. Synthesis of optically complex core-shell colloidal suspensions: Pathways to multiplexed biological screening. Adv Funct Mat 13(11): 887-896. University of Texas, Austin Harvey, B.R., G. Georgiou, A. Hayhurst, K.J. Jeong, B.L. Iverson, and G.K. Rogers. 2004. Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. PNAS 101(25): 9193-9198. Kawarasaki, Y., K.E. Griswold, J.D. Stevenson, T. Selzer, S.J. Benkovic, Iverson, B.L., and G. Georgiou. 2003. Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple nonhomologous crossovers. Nucleic Acids Res 31(21): e126. University of Zurich Sirena, D., B. Lilienfeld, M. Eisenhut, S. Kälin, K. Boucke, R.R. Beerli, L. Vogt, C. Ruedl, M.F. Bachmann, U.F. Greber, and S. Hemmi. 2004. The Human Membrane Cofactor CD46 Is a Receptor for Species B Adenovirus Serotype 3. J Virol 78(9): 4454-4462. Stanford University Wehrman, T., B. Kleaveland, J.Her., R.F. Balint, and H.M. Blau. 2002. Protein-protein interactions monitored in mammalian cells via complementation of b-lactamase enzyme fragments. PNAS 99(6): 3469-3474. 1. Ashcroft, R.G., and P.A. Lopez. 2000. Commercial high speed machines open new opportunities in high throughput flow cytometry. J Immunol Methods 243: 13. 2. Battye, F.L., A. Light, and D.M. Tarlinton. 2000. Single cell sorting and cloning. J Immunol Methods 243: 25. 3. Daugherty, P.S., B.L. Iverson, and G. Georgiou. 2000. Flow cytometric screening of cell-based libraries. J mmunol Methods 243: 211. 138 CyAn ADP with Summit Software User Guide

MHC multimer assays Fluorescent major histocompatibility (MHC) class I- and class II-peptide multimers are finding increasing application in the study of antigen-specific T cells. Together, the CyAn ADP High- Perfomance Analyzer and the MoFlo High-Performance Cell Sorter allow rapid detection and purification of these cells, which typically comprise less than 1% of peripheral blood mononuclear cells. CyAn ADP and MoFlo Memorial Sloan-Kettering Cancer Center Dao, T., D. Guo, A. Ploss, A. Stolzer, C. Saylor, T.E. Boursalian, J.S. Im, D.B. Sant Angelo. 2004. Development of CD1drestricted NKT cells in the mouse thymus. Eur J Immunol 34(12): 3542-3552. Technical University of Munich Huster, K.M., V. Busch, M. Schiemann, K. Linkemann, K.M. Kerksiek, H. Wagner, and D.H. Busch. 2004. Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. PNAS 101(15): 5610-5615. University of Pittsburgh School of Medicine Meidenbauer, N., T.K. Hoffmann, A.D. Donnenberg. 2003. Direct visualization of antigen-specific T cells using peptide- MHC-class I tetrameric complexes. Methods 31(2): 160-171. CyAn ADP Trudeau Institute Mayer, K.D. Mohrs, K., S.R. Crowe, L.L. Johnson, P. Rhyne, D.L. Woodland, and M. Mohrs. 2005. The functional heterogeneity of type 1 effector T cells in response to infection is related to the potential for IFN-gamma production. J Immunol. 2005. 174(12): 7732-7739. Kemp, R.A., T.J. Powell, D.W. Dwyer, R.W. Dutton. 2004. Cutting edge: Regulation of CD8+ T cell effector population size. J. Immunol 173(5): 2923-2927. MoFlo Basel Institute for Immunology Bachmann, M.F., A. Gallimore, S. Linkert., V. Cerundolo, A. Lanzavecchia, M. Kopf, and A. Viola. 1999. Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naïve and memory T cells. J Exp Med 189(10): 1521-1529. Harvard Medical School Stratmann, T., N. Martin-Orozco, V. Mallet-Designe, L. Poirot, D. McGavern, G. Losyev, C.M. Dobbs, M.B.A. Oldstone, K. Yoshida, H. Kikutani, D. Mathis, C. Benoist, K. Haskins, and L. Teyton. 2003. Susceptible MHC alleles, not background genes, select an autoimmune T cell reactivity. J Clin Invest 112(6): 902-914. Memorial Sloan-Kettering Cancer Center Koehne, G., K.M. Smith, T.L Ferguson, R. Y. Williams, G. Heller, E.g. Pamer, B. Dupont, and R. O Reilly. 2002. Quantitation, selection and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon-γ production and growth of cytotoxic precursors. Blood 99(5): 1730-1740. Koehne, G., H.F. Gallardo, M. Sadelain, and R.J. O Reilly. 2000. Rapid selection of antigen-specific T lymphocytes by retroviral transduction. Blood 96(1): 109-117. CyAn ADP with Summit Software User Guide 139

New York University School of Medicine Mucida, D., N. Kutchukhidze, A. Erazo, M. Russo, J.J. Lafaille, M.A. Curotto de Lafaille. 2005. Oral tolerance in the absence of naturally occurring Tregs. J Clin Invest prepublished online. St. Jude Children s Research Hospital Marshall, D.R., E. Olivas, S. Andreansky, N.L. La Gruta, G.A. Neale, A. Gutierrez, D.G. Wichlan, S. Wingo, C. Cheng, P.C. Doherty, and S.J. Turner. 2005. Effector CD8+ T cells recovered from an influenza pneumonia differentiate to a state of focused gene expression. PNAS 102(17): 6074-6079. Kedzierska, K., S.J. Turner, and P.C. Doherty. 2004. Conserved T cell receptor usage in primary and recall responses to an immunodominant influenza virus nucleoprotein epitope. PNAS 101(14): 4942-4947. Turner, S. J., R. Cross, W. Xie, and P.C. Doherty. 2001. Concurrent naive and memory CD8+ T cell responses to an influenza A virus. J Immunol 167(5): 2753-2758. South Carolina Cancer Center Szmania, S., A. Galloway, M. Bruorton, P. Musk, G. Aubert, A. Arthur, H. Pyle, N. Hensel, N. Ta, L. Lamb, T. Dodi, A. Madrigal, J. Hensell-Downey, and F. van Rhee. 2001. Isolation and expansion of cytomegalovirus-specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA-tetramers. Blood 98(3): 505-512. University of California, Davis Kita, H., S. Matsumura, S. He, A.A. Ansari, Z. Lian, J. Van de Water. R.l. Coppel, M.M. Kaplan, and M.E. Gershwin. 2002. Quantitative and functional analysis of PDC-E2-specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis. J Clin Investigation 109(9): 1231-1240. University of Chicago Chun, T., M.J. Page, L. Gapin, J.L. Matsuda, H. Xu, H. Nguyen, H.S. Kang, A.K. Stanic, S. Joyce, W.A. Koltun, M.J. Chorney, M. Kronenberg, and C.R. Wang. 2003. CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells. J Exp Med 197(7): 907-918. University of North Carolina Wang, B., A. Sharma, R. Maile, M. Saad, E.J. Collins, and J.A. Frelinger. 2002. Peptidic termini play a significant role in TCR recognition. J Immunol 169: 3137-3145. University of Pennsylvania Cancer Center Maus, M.V., B. Kovacs, W.W. Kwok, G.T. Nepom, K. Schlienger, J.L. Riley, D. Allman, T.H. Finkel, and C.H. June. 2004. Extensive replicative capacity of human central memory T cells. J Immunol 172(11): 6675-6683. Vonderheide, R.H., S.M. Domchek, J.L. Schultze, D.J. George, K.M. Hoar, D.-Y. Chen, K.F. Stephans, K. Masutomi, M. Loda, Z. Xia, K.S. Anderson, W.C. Hahn, and L.M. Nadler. 2004. Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes. Clin Cancer Res 10(3): 828-839. Maus, M., A.K. Thomas, D.G.B. Leonard, D. Allman, K. Addya, K. Schlienger, J.L. Riley, and C.H. June. 2002. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nature Biotechnol 20: 143-148. 140 CyAn ADP with Summit Software User Guide

Stem cells Both hematopoietic and non-hematopoietic stem cells continue to garner increasing attention in laboratories around the world. Studies continue to define phenotypic markers, in vivo reconstitutional activity, and creation of model systems for stem cell development, multi-drug resistance (mdr) and use in genetic therapies. Together, the CyAn ADP High-Performance Analyzer and the MoFlo High-Performance Cell Sorter are ideal for identifying, isolating and recovering viable populations of these valuable cells from a variety of sample sources. CyAn ADP and MoFlo University of Pittsburgh School of Medicine Yuan, Y., H. Shen, D.S. Franklin, D.T. Scadden, T. Cheng. 2004. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18ink4c. Nature Cell Biol 6(5): 436-442. CyAn ADP John Radcliffe Hospital Sternberg, A., S. Killick, T. Littlewood, C. Hatton, A. Peniket, T. Seifl, S. Soneji, J. Leach, D. Bowen, C. Chapman, E. Massey, G. Standen, L. Robinson, B. Vadher, R. Kaczmarski, R. Janmohammed, K. Clipsham, A. Carr, and P. Vyas. 2005. Evidence for reduced B-cell progenitors in early (low-risk) myelodysplastic syndrome. Blood 106(9): 2982-2991. Oxford University North, T.E., T. Stacy, C.J. Matheny, N.A. Speck, M.F.T.R. de Bruijn. 2004. Runx1 is expressed in adult mouse hematopoietic stem cells and differentiating myeloid and lymphoid cells, but not in maturing erythroid cells. Stem Cells 22: 158-168. University of Pittsburgh School of Medicine Yuan, Y., H. Yu, M. Boyer, X. Song, S. Cao, H. Shen, and T. Cheng. 2005. Hematopoietic stem cells are not the direct target of spontaneous leukemic transformation in p18ink4c-null reconstituted mice. Cancer Research 66(1): 343-351. University of York Etheridge, S.L., G.J. Spencer, D.J. Heath, P.G. Genever. 2004. Expression Profiling and functional analysis of Wnt signaling mechanisms in mesenchymal stem cells. Stem Cells 22(5): 849-860. MoFlo Basel Institute for Immunology Schaniel, C., L. Bruno, F. Melchers, and A.G. Rolink. 2002. Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5-deficient pre-b I-cell clones. Blood 99: 472. Baylor College of Medicine Hirschmann-Jax, C., A.E. Foster, G.G. Wulf, J.G. Nuchtern, T.W. Jax, U. Gobel, M.A. Goodell, and M.K. Brenner. 2004. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. PNAS 101(39): 14228-14233. Majka, S.M., K.A. Jackson, K.A. Kienstra, M.W. Majesky, M.A. Goodell, and K.K. Hirschi. 2003. Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration. J Clin Invest 111: 71. McKinney-Freeman, S.L., K.A. Jackson, F.D. Camargo, G. Ferrari, F. Mavilio, and M.A. Goodell. 2002. Muscle-derived hematopoietic stem cells are hematopoietic in origin. PNAS 99: 1341. Jackson, K.A., S. M. Majka, H. Wang, J. Pocius, C.J. Hartley, M. W. Majesky, M.L. Entman, L.H. Michael, K.K. Hirschi, and M.A. Goodell. 2001. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 107: 1395. CyAn ADP with Summit Software User Guide 141

Kuehnle, I., M.H. Huls, Z. Liu, M. Semmelmann, R.A. Krance, M.K. Brenner, C.M. Rooney, and H.E. Heslop. 2000. CD20 monoclonal antibody (rituximab) for therapy of Epstein-Barr virus lymphoma after hematopoietic stem-cell transplantation. Blood 95: 1502. Jackson, K.A., T. Mi, and M.A. Goodell. 1999. Hematopoietic potential of stem cells isolated from murine skeletal muscle. PNAS 96: 14482. Brigham and Women s Hospital Dimitroff, C.J., J.Y. Lee, R.C. Fuhlbrigge, and R. Sackstein. 2000. A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells. PNAS 97(25): 13841. City of Hope National Medical Center Shih, C.C., Y. Weng, A. Mamelak, T. LeBon, M.C. Hu, and S.J. Forman. 2001. Identification of a candidate human neurohematopoietic stem-cell population. Blood 98: 2412. Shih, C.C., M.C. Hu, J. Hu, Y. Weng, P.J. Yazaki, J. Medeiros, and S.J. Forman. 2000. A secreted and LIF-mediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells. Blood 95: 1957. Chatterjee, S., W. Li, C.A. Wong, G. Fisher-Adams, D. Lu, M. Guha, J.A. Macer, S.J. Forman, and K.K. Wong, Jr. 1999. Transduction of primitive human marrow and cord blood-derived hematopoietic progenitor cells with adeno-associated virus vectors. Blood 93: 1882. Shih, C.C., M.C. Hu, J. Hu, J. Medeiros, and S.J. Forman. 1999. Long-term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells. Blood 94: 1623. Dana-Farber Cancer Institute Hock, H., M.J. Hamblen, H.M. Rooke, J.W. Schindler, S. Saleque, Y. Fujiwara, and S.H. Orkin. 2004. Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells. Nature 431(7011): 1002-1007. Mikkola, H.K.A., Y. Fujiwara, T.M. Schlaeger, D. Traver, and S.H. Orkin. 2003. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood 101: 508. Okuno, Y., H. Iwasaki, C.S. Huettner, H.S. Radomska, D.A. Gonzalez, D.G. Tenen, and K. Akashi. 2002. Differential regulation of the human and murine CD34 genes in hematopoietic stem cells. PNAS 99: 6246. Okuno, Y., C.S. Huettner, H.S. Radomska, V. Petkova, H. Iwasaki, K. Akashi, and D.G. Tenen. 2002. Distal elements are critical for human CD34 expression in vivo. Blood 100: 4420. Radomska, H.S., D.A. Gonzalez, Y. Okuno, H. Iwasaki, A. Nagy, K. Akashi, D.G. Tenen, and C.S. Huettner. 2002. Transgenic targeting with regulatory elements of the human CD34 gene. Blood 100: 4410. European Molecular Biology Laboratory Musaro, A., C. Giacinti, G. Borsellino, G. Dobrowolny, L. Pelosi, L. Cairns, S. Ottolenghi, G. Cossu, G. Bernardi, L. Battistini, M. Molinaro, and N. Rosenthal. 2004. Stem cell-mediated muscle regeneration is enhanced by local isoform of insulin-like growth factor 1. PNAS 101(5): 1206-1210. Harvard University Dor, Y., J. Brown, O.I. Martinez, D.A. Melton. 2004. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429: 41-46 Haukeland University Hospital Tsinkalovsky, O., B. Rosenlund, O.Didrik Laerum, and H. Geir Eiken. 2005. Clock gene expression in purified mouse hematopoietic stem cells. Exp Hematol 33(1): 100-107. Mt. Sinai School of Medicine Huber, T. L., V. Kouskoff, H. Joerg Fehling, J. Palis, G. Keller. 2004. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 432(7017): 625-630. 142 CyAn ADP with Summit Software User Guide

Henckaerts, E., H. Geiger, J.C. Langer, P. Rebollo, G. Van Zant, and H.W. Snoeck. 2002. Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to earlyacting cytokines. Blood 99: 3947. Ottawa Health Research Institute Asakura, A., P. Seale, A. Girgis-Gabardo, and M.A. Rudnicki. 2002. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 159: 123. Roger Williams Medical Center Mogi, M., J. Yang, J.-F. Lambert, G.A. Colvin, I. Shiojima, C. Skurk, R. Summer, A. Fine, P.J. Quesenberry, and K. Walsh. 2003. Akt signaling regulates side population cell phenotype via Bcrp1 translocation. J Biol Chem 278(40): 39068-39075. Stanford University School of Medicine Lu, L. S., J. Tung, N. Baumgarth, O. Herman, M. Gleimer, and L.A. Herzenberg. 2002. Identification of a germ-line pro-b cell subset that distinguishes the fetal/neonatal from the adult B cell development pathway. PNAS 99: 3007. Manz, M.G., T. Miyamoto, K. Akashi, and I.L. Weissman. 2002. Prospective isolation of human clonogenic common myeloid progenitors. PNAS 99: 11872-11877. Wright, D.E., E.P. Bowman, A.J. Wagers, E.C. Butcher, and I.L. Weissman. 2002. Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. J Exp Med 195: 1145. Stowers Institute for Medical Research Akashi, K., X. He, J. Chen, H. Iwasaki, C. Niu, B. Steenhard, J. Zhang, J. Haug, and L. Li. 2003. Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis. Blood 101: 383. University of California, Davis Chute, J.P., G.G. Muramoto, J. Fung, and C. Oxford. 2005. Soluble factors elaborated by human brain endothelial cells induce the concomitant expansion of purified human BM CD34+CD38 cells and SCID-repopulating cells. Blood 105(2): 576-583. University of Cambridge Campos, L.S., D.P. Leone, J.B. Relvas, C. Brakebusch, R. Fässler, U. Suter, and C. ffrench-constant. 2004. Beta-1 integrins activate a MAPK signalling pathway in neural stem cells that contributes to their maintenance. Devel 131(14): 3433-3444. University of Colorado Health Sciences Center Pyatt, D.W., W.S. Stillman, Y. Yang, S. Gross, J.H. Zheng, and R.D. Irons. 1999. An essential role for NF-κB in human CD34+ bone marrow cell survival. Blood 93: 3302. University of Freiburg Medical Center Goan, S.R., I. Junghahn, M. Wissler, M. Becker, J. Aumann, U. Just, G. Martiny-Baron, I. Fichtner, and R. Henschler. 2000. Donor stromal cells from human blood engraft in NOD/SCID mice. Blood 96: 3971. Spyridonidis, A., M. Schmidt, W. Bernhardt, A. Papadimitriou, M. Azemar, W. Wels, B. Groner, and R. Henschler. 1998. Purging of mammary carcinoma cells during ex vivo culture of CD34+ hematopoietic progenitor cells with recombinant immunotoxins. Blood 91: 1820. CyAn ADP with Summit Software User Guide 143

University of Massachusetts Medical School Hulspas, R., and P.J. Quesenberry. 2000. Characterization of neurosphere cell phenotypes by flow cytometry. Cytometry 40: 245-250. Habibian, H.K., S.O. Peters, C.C. Hsieh, J. Wuu, K. Vergilis, C.I. Grimaldi, J. Reilly, J.E. Carlson, A.E. Frimberger, F.M. Stewart, and P.J. Quesenberry. 1998. The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. J Exp Med 188: 393. Stewart, F.M., S. Zhong, J. Wuu, C. Hsieh, S.K. Nilsson, and P.J. Quesenberry. 1998. Lymphohematopoietic engraftment in minimally myeloablated hosts. Blood 91(10): 3681-3687. University of North Carolina, Chapel Hill Kirby, S., W. Walton, and O. Smithies. 2000. Hematopoietic stem cells with controllable tepor transgenes have a competitive advantage in bone marrow transplantation. Blood 95: 3710. Kubota, H., and L.M. Reid. 2000. Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. PNAS 97: 12132. University of Pennsylvania School of Medicine Izon, D.J., J.C. Aster, Y. He, A. Weng, F.G. Karnell, V. Patriub, L. Xu, S. Bakkour, C. Rodriguez, D. Allman, and W.S. Pear. 2002. Deltex1 redirects lymphoid progenitors to the B cell lineage by antagonizing Notch1. Immunity 16: 231. University of Southern California Zhong, J.F., Y. Zhao, S. Sutton, A. Su, Y. Zhan, L. Zhu, C. Yan, T. Gallaher, P.B. Johnston, W.F. Anderson, and M.P. Cooke. 2005. Gene expression profile of murine long-term reconstituting vs. short-term reconstituting hematopoietic stem cells. PNAS 102(7): 2448-2453. Zhong, J.F., Y. Zhan, W.F. Anderson, and Y. Zhao. 2002. Murine hematopoietic stem cell distribution and proliferation in ablated and nonablated bone marrow transplantation. Blood 100: 3521. Walter and Eliza Hall Institute Metcalf, D., L. Di Rago, and S. Mifsud. 2002. Synergistic and inhibitory interactions in the in vitro control of murine megakaryocyte colony formation. Stem Cells 20: 552. Ogilvy, S., D. Metcalf, L. Gibson, M.L. Bath, A.W. Harris, and J.M. Adams. 1999. Promoter elements of vav drive transgene expression in vivo throughout the hematopoietic compartment. Blood 94: 1855. Washington University School of Medicine Xian, H.Q., E. McNichols, A. St. Clair, D.I. Gottlieb. 2003. A subset of ES-cell derived neural cells marked by gene targeting. Stem Cells 21: 41-49. 144 CyAn ADP with Summit Software User Guide

T- and B-cells Classic subjects for flow cytometric analysis, T and B lymphocytes play a central role in the function of the immune system. With their powerful and adaptive platforms, the CyAn ADP High- Performance Analyzer and the MoFlo High-Performance Cell Sorter enable efficient multiparametric identification and isolation of the almost infinite number of subsets of these and other immune cell types, revealing the complexities of cellular and humoral immune response. CyAn ADP and MoFlo Memorial Sloan-Kettering Cancer Center Dao, T., D. Guo, A. Ploss, A. Stolzer, C. Saylor, T.E. Boursalian, J.S. Im, and D.B. Sant Angelo. 2004. Development of CD1d-restricted NKT cells in the mouse thymus. Eur J Immunol 34(12): 3542-3552. National Jewish Medical and Research Center Dal Porto, J.M., K. Burke, and J.C. Cambier. 2004. Regulation of BCR signal transduction in B-1 cells requires the expression of the Src family kinase Lck. Immunity 21: 443-453. Maier, H., R. Ostraat, H. Gao, S. Fields, S.A. Shinton, K.L. Medina, T. Ikawa, C. Murre, H. Singh, R.R. Hardy, J. Hagman. 2004. Early B cell factor cooperates with Runx1 and mediates epigenetic changes associated with mb-1 transcription. Nature Immunol 5(10): 1069-1077. University of Georgia Martin, D.L. and R.L. Tarleton 2005. Antigen-specific T cells maintain an effector memory phenotype during persistent Trypanosoma cruzi infection. J Immunol 174: 1594-1601. University of California San Francisco School of Medicine Lohr, J., B. Knoechel, E.C. Kahn, and A.K. Abbas. 2004. Role of B7 in T cell tolerance. J Immunol 173(8): 5028-5035. Lohr, J., B. Knoechel, S. Jiang, A.H. Sharpe, A.K. Abbas. 2003. The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens. Nature Immunol 4(7): 664-669. CyAn ADP John Radcliffe Hospital Amyes, E., A. McMichael, and M. Callan. 2005. Human CD4+ T Cells are predominantly distributed among six phenotypically and functionally distinct subsets. J. of immunol 175(9): 5765-5773. Memorial Sloan-Kettering Cancer Center Kim, H., D. Guo, and D.B. Sant Angelo. 2005. Coevolution of TCR-MHC interactions: Conserved MHC tertiary structure is not sufficient for interactions with the TCR. PNAS 102(20): 7263-7267. Technical University of Munich Yu, P., R. Constien, N. Dear, M. Katan, P. Hanke, T. Bunney, S. Kunder, L. Quintanilla-Martinez, U. Huffstadt, A. Schroder, N. Jones, T. Peters, H. Fuchs, M. Hrabe de Angelis, M. Nehls, J. Grosse, P. Wabnitz, T. Meyer, K. Yasuda, M. Schiemann, C. Schneider-Fresenius, W. Jagla, A. Russ, A. Popp, M. Josephs, A. Marquardt, J. Laufs, C. Schmittwolf, H. Wahner, K. Pfeffer, G. Mudde. 2005. Autoimmunity and inflammation due to a gain of function mutation in phospholipase Cy2 that specifically increased external Ca 2+ entry. Immunity 22(4):451-465. Trudeau Institute André Gessner, K. Mohrs, and M. Mohrs. 2005. Mast cells, basophils, and eosinophils acquire constitutive IL-4 and IL-13 transcripts during lineage differentiation that are sufficient for rapid cytokine production. J of Immunol 174 (2): 1063-1072. Mohrs, K, D. Harris, F. Lund, M. Mohrs. 2005. Systemic dissemination and persistence of Th2 and type 2 cells in response to infection with a strictly enteric nematode parasite. J of Immunol 175 (8): 5306-5313. Wistar Institute CyAn ADP with Summit Software User Guide 145

Papasavvas, E., J. Sun, Q. Luo, E. Moore, B. Thiel, R. MacGregor, A. Minty, K. Mounzer, J. Kostman, and L. Montaner. 2005. IL-13 acutely augemtns HIV-specific and recall responses from HIV-1 infected subjects in vitro by modulating monocytes. J. of immunol 175(8): 5532-5540. MoFlo GKT Medical School, Guy s Campus Pennington, D. J., B. Silva-Santos, J. Shires, E. Theodoridis, C. Pollitt, E.L Wise, R.E Tigelaar, M.J. Owen, and A.C. Hayday. 2003. The inter-relatedness and interdependence of mouse T cell receptor gamma delta and alpha beta cells. Nature Immunol 4: 991-998. Girardi, M., D.E. Oppenheim, C.R. Steele, J.M. Lewis, E. Glusac, R. Filler, P. Hobby, B. Sutton, R.E. Tigelaar, and A.C. Hayday. 2001. Regulation of cutaneous malignancy by gamma-delta T cells. Science 294(5542): 605-609. Harvard Medical School Tager, A. M., S. K. Bromley, B.D. Medoff, S.A. Islam, S.D. Bercury, E.B. Friedrich, A.D. Carafone, R.E. Gerszten, and A.D. Luster. 2003. Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nature Immunol 4: 982-990. Sánchez-Fueyo, A., J. Tian, D. Picarella, C. Domenig, X.X. Zheng, C.A Sabatos, N. Manlongat, O. Bender, T. Kamradt, V.K. Kuchroo, J.-C. Gutiérrez-Ramos, A.J. Coyle, and T.B. Strom. 2003. Tim-3 inhibits T helper type 1 mediated autoand alloimmune responses and promotes immunological tolerance. Nature Immunol 4: 1093-1101. Institut Pasteur Vohenrich, C.A.J., A. Cumano, W. Müller, J.P. Di Santo, and P. Vieira. 2003. Thymic stromal-derived lymphopoietin distinguishes fetal from adult B cell development. Nature Immunol 4: 773-779. IRCCS Santa Lucia Angelini, D.F., G. Borsellino, M. Poupot, A. Diamantini, R. Poupot, G. Bernardi, F. Poccia, J.-J. Fournie, and L. Battistini. 2004. Fc{gamma}RIII discriminates between 2 subsets of V{gamma}9V{delta}2 effector cells with different responses and activation pathways. Blood 104(6): 1801-1807. National Jewish Medical and Research Center Oliver, P.M., M. Wang, Y. Zhu, J. White, J. Kappler, and P. Marrack. 2004. Loss of Bim allows precursor B cell survival but not precursor B cell differentiation in the absence of Interleukin 7. J Exp Med 200(9): 1179-1187. Ott, V.L., J.C. Cambier, J. Kappler, P. Marrack, B.J. Swanson. 2003. Mast cell dependent migration of effector CD8+ T cells through production of leukotriene B4. Nature Immunol 4: 974-981. Teague, T.K., D. Hildeman, R.M. Kedl, T. Mitchell, W. Rees, B.C. Schaefer, J. Bender, J. Kappler, and P. Marrack. 1999. Activation changes the spectrum but not the diversity of genes expressed by T cells. PNAS 96(22): 12691-6. Ontario Cancer Institute Fleming, H.E., C.D. Milne, and C.J. Paige. 2004. CD45-deficient mice accumulate pro-b cells both in vivo and in vitro. J Immunol 173(4): 2542-2551. RIKEN Research Center, Kanagawa Kamimura, D., N. Ueda, Y. Sawa, S. Hachida, T. Atsumi, T. Nakagawa, S. Sawa, G.-H. Jin, H. Suzuki, K. Ishihara,, M. Murakami, and T. Hirano. 2004. Evidence of a novel IL-2/15R{beta}-targeted cytokine involved in homeostatic proliferation of memory CD8+ T cells. J Immunol 173(10): 6041-6049. 146 CyAn ADP with Summit Software User Guide

Université de Montréal Priceputu, E., I. Rodrigue, P. Chrobak, J. Poudrier, T.W. Mak, Z. Hanna, C. Hu, D.G. Kay, and P. Jolicoeur. 2005. The Nef-mediated AIDS-like disease of CD4C/human immunodeficiency virus transgenic mice is associated with increased Fas/FasL expression on T cells and T-cell death but Is not prevented in Fas-, FasL-, tumor necrosis factor receptor 1-, or interleukin-1{beta}-converting enzyme-deficient or Bcl2-expressing transgenic mice. J Virol 79(10): 6377-6391. Weng, X., E. Priceputu, P. Chrobak, J. Poudrier, D.G. Kay, Z. Hanna,T.W. Mak, and P. Jolicoeur. 2004. CD4+ T cells from CD4C/HIVNef transgenic mice show enhanced activation In vivo with impaired proliferation in vitro but are dispensable for the development of a severe AIDS-like organ disease. J Virol 78(10): 5244-5257. University of Alabama, Birmingham Pancer, Z., C.T. Amemiya, G.R.A. Ehrhardt, J. Ceitlin, G.L. Gartland, and M.D. Cooper. 2004. Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 430(6996): 174-180. University of California, San Francisco Matloubian, M., C.G. Lo, G. Cinamon, M.J.Lesneski, Y. Xu, V. Brinkmann, M.L. Allende, R.L. Proia, and J.G. Cyster. 2004. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427: 355-360. Davies, S.J., J.L. Grogan, R.B. Blank, K.C. Lim, R.M. Locksley, and J.H. McKerrow. 2001. Modulation of blood fluke development in the liver by hepatic CD4+ lymphocytes. Science 294(5545): 1358-1361. University of Pennsylvania Cancer Center Mullen, A.C., F.A. High, A.S. Hutchins, H.W. Lee, A.V. Villarino, D.M. Livingston, A.L. Kung, N. Cereb, T.-P. Yao, S.Y. Yang, and S.L. Reiner. 2001. Role of T-bet in commitment of TH1 cells before IL-12-dependent selection. Science 292(5523): 1907-1910. Washington University School of Medicine Kemper, C., A.C. Chan, J.M. Green, K.A. Brett, K.M. Murphy, and J.P. Atkinson. 2003. Activation of human CD4+ cells with CD3 and CD46 induces a T-regulatory cell 1 phenotype. Nature 421: 388-392. CyAn ADP with Summit Software User Guide 147