FACS Laboratory. BD LSRs Operator Training. Training Guide.

Transcription

1 FACS Laboratory Training Guide BD LSRs Operator Training

2 LRI FACS lab

3 1 Components 1.1 Basic parts overview.pg3-6 Overview of LSRs. 3 Filling and emptying the tanks Starting up the LSR...6 Cleaning and Shutting down Fluidics.pg7-11 Basic fluidic mechanics in flow cytometry 7 The sample injection port (SIP)..9 Troubleshooting.10 o Unblocking a probe 10 o De-bubbling filters Optics...pg12-31 Optics Overview. 12 Detection optics Digital electronics pg32-34 Digital flow cytometry and the LSRs.32 Digital pulse processing Fluorochromes & Multi-colour Flow 2.1 Commonly used fluorochromes..pg Fluorochrome selection Viability dyes..39 Fixable LIVE/DEAD amine reactive dyes.40 Availability 40 Physical Characteristics Combining fluorochromes.pg41-43 Spectral overlap 41 Problem pairs. 41 Example combinations 42

4 2.4 Compensation.pg44-49 Controls. 44 Compensation beads. 45 The bi-exponential display. 47 Compensation - the median fluorescence technique.. 37 Copying spectral overlap. 49 Automatic compensation BD FACSDiva TM : Software Basics 3.1 The Browser, Worksheet, Inspector and Acquisition windows..pg Example experiment.pg52-73 Working with plots, regions and gates. 55 Baseline PMT voltages 62 Compensation 63 o Automatic compensation...68 Exporting data from FACSDiva as listmode (.fcs) files 72 Working with templates 73 4 Quality Control 4.1 Routine Checks.. pg Laser Delay, Area Scaling and Windows Extension.pg Sample preparation 5.1 Harvesting cells.pg Resuspending cells for analysis pg78 6 Glossary. pg Troubleshooting Guide. pg85

5 1.1 Basic Parts Overview In the FACS Lab, all cytometers are normally switched on at 9am. When booking to start before or at 9, or on weekends, please be aware that the LSRs have multiple laser lines that need to be given a minimum of 30 minutes to warm up before running samples. Overview of LSRs From the control panel you can perform the operations RUN, STANDBY and PRIME, and make some sample flow rate adjustments. The machine should be on STANDBY with a tube of water when you come to it. Check that the sheath tank is filled to the line and the waste tank is empty. If the sheath tank runs dry, air will enter the system and can be difficult to remove; you will be unable to acquire any data if this happens. * Important! * Before starting an experiment, check the levels of sheath fluid and waste. If you are using the machine for a long time check the tanks regularly. We cannot stress enough how critical it is that you are capable of maintaining the tanks and purging air from the system. If you are unsure of how to do so, please ask a member of the FACS Lab, to avoid ruining an important experiment, and possibly the next user s experiment too! LSRIIA and LSRIIB overview 1. Power switch 2. Sheath tank 3. Waste tank 4. Control panel 5. Sample Injection Port (SIP) 6. Sheath Filter 7. Sheath line air purge (roll-valve) Fig Location of important parts on LSRII. 3

6 LSRFortessa A, B and C overview 1. Power switch 2. Sheath tank 3. Waste tank 4. Control panel 5. Sample Injection Port (SIP) 6. Sheath filter 7. Sheath line air purge (roll-valve) Fig Location of important parts on LSRFortessa 4

7 Filling and emptying the tanks A B Fig A De-pressurising sheath tank by pulling up on valve (black circle). B Lid of waste tank showing waste line (orange tube) and alarm (black wire). First check the machine is in STANDBY (standby button on control panel is orange): *Filling the sheath tank* Fig A de-pressurising tank Unclip the sheath pressure line (red circle - green tube on top of tank). De-pressurise the tank (black circle - pull metal release valve up). Unscrew & remove lid. Fill with PBS to groove in tank - do not overfill. PBS can be located in bottles on the shelf in the corridor. Replace the lid and re-connect the pressure line. De-bubble the LSR it is good practice to always do so when you re-fill the tank. Please refer to the Troubleshooting section in section 1.2 for instructions. *Emptying the waste* Fig B waste tank lid Unclip the orange line (waste line) and disconnect the alarm (black wire - twist connection) from the lid of the waste tank. Unscrew the lid: add 100g Virkon powder (use measuring cup in Virkon container) to the tank. Empty the waste into the sink by LSRIIA (Virkon is kept under the sink). Replace lid, re-connect waste line and alarm. 5

8 Starting up the LSR Switch on the LSR and start the computer workstation. There is no password to log into Windows (hit Enter ). Priming the machine Before running any samples it is good practice to prime the fluidics to remove any bubbles from the flow cell. Additionally, before you run your sample (or while you are waiting for the lasers to warm up), it is a good idea to check the machine is clean by simply running fresh dh 2 0 in a new tube. If move arm to side during Prime, remember to return it after! When running dh20, have threshold at 15,000 and FSC at voltage used for cells. The event rate should be mostly 0 events/second but up to 100evt over 30 seconds due to bubbles, noise. If you have time, it does not hurt to run some cleaning agents for a short period, followed by dh 2 0. After these steps and allowing the lasers to warm up, the machine is ready to run your samples - this will be covered later. *Priming the LSR* Remove the tube of dh 2 0 from the sample injection port (SIP). With SIP arm to the side, press PRIME; this empties out the flow cell and refills it to remove bubbles. LSR returns to STANDBY when done. Repeat 1-2 times. Cleaning and shutting down When you are done running your samples, it is imperative to clean the LSR sufficiently to prevent clogging of the SIP and remove dyes which may remain in the tubing: *Cleaning procedure* Run tube of detergent for 5 min on HI (BD FACS Rinse). Run tube of bleach (BD FACS Clean) for 3min on HI if DNA dyes/viability dye such as DAPI, Hoechst, PI etc. have been used; if not - proceed to next step. Run tube of dh 2 0 for 5min. Check the machine is clean with a fresh tube of dh 2 0. If machine is not clean, repeat Clean & Rinse steps. If the machine has been left dirty, running cleaning agents for a longer length of time will usually help; otherwise the machine may require a long clean which is routinely performed by the FACS Lab staff. The last user of the day should switch off the cytometer after the cleaning. IMPORTANT! Read also Troubleshooting, in Section purging air from the machine. 6

9 1.2 Fluidics A pressurised fluidics system delivers the sample to the flow cell where interrogation by the lasers takes place, using the process of hydrodynamic focusing to help create a tight, single cell flow to allow us to look at each cell one by one. This is achieved by laminar flow, higher sheath to core velocities and by the shape of the flow cell. Lasers Flow cell Sample Vacuum Waste Sheath Lo/Med/Hi controls Pressure line Fig Basic diagram of flow cytometer fluidics system Basic Fluid Mechanics in Flow Cytometry In the cuvette type cytometers (e.g. Caliburs, LSRs), cells are introduced into the middle of the flow cell, injected into the centre of a smoothly flowing stream (the sheath fluid). Since both the core stream (consisting of your injected sample) and the sheath stream are flowing smoothly, laminar flow is observed where the two will maintain their relative positions and do not mix. We want to analyse one cell at a time, so a narrow core carrying single cells to the laser interrogation point(s) is critical. One of the ways this is achieved is by using a cuvette where the sides taper inwards towards the area of laser interrogation. 7

10 Note that fluid flow at the exit from the region of cuvette constriction into the narrower region where laser interrogation occurs can be described as slug flow (having constant velocity across the fluid cross section). This continues for a while, but quickly returns to a parabolic laminar flow profile (velocity at the edges is slower than at the centre). Fig Fluid dynamics in the flow cell. (Shapiro s Practical Flow Cytometry, 4th Ed.) It is important that laser interrogation occurs before the parabolic profile is reestablished so that cells will have the same velocity - and therefore take the same time to pass through the laser beam, regardless of the position within the core. In other words, variations in distribution will not have an impact on illumination time, giving better instrument precision. (Further detail can be found in Howard Shapiro s Practical Flow Cytometry 4 th Ed). Which speed to run your sample- HI, MED, LO? Although we cannot adjust the pressure of the sheath, we can change the pressure applied to the sample, to inject more/less sample per unit of time. This is useful when the sample is very dilute or to save time when looking for very rare events (i.e. need to run through a large number of cells). Increasing the sample pressure will result in a widening of the core carrying your particles of interest, as shown in Fig This affects how uniformly particles flow past the interrogation points and will affect measurements such as DNA (which are best run in LO) where precision is important. Increasing sample pressure will result in an increase in DNA histogram CV. For qualitative measurements such as immuno-phenotyping, there may be some loss of resolution but running in MED/HI is generally not a problem, when using log scale. 8

11 Fig Core stream diameter increase is observed when sample pressure is high (right) compared to low sample pressure (left). Samples should be sufficiently concentrated to enable a reasonable run time, especially if you need to run your samples in LO. Correct sample preparation (see section 5) is also vital to prevent blocking the probe: there is no PAUSE function in DIVA, one must stop recording, clear the blockage, then APPEND to the saved file. It is better to avoid having clumps in the first place. The sample injection port (SIP) The SIP comprises the sample injection tube and support arm. The sample injection tube is encased in a sleeve. When the support arm is open, the sample injection tube backflushes sheath fluid. This is then removed by a vacuum pump (activated when the arm is open) up the outer sleeve to the waste tank, helping to clean the injection tube between samples. Fig Sample injection port component diagram and photograph. 9

12 *Important!* The machine starts taking up your sample as soon as you have installed the tube on the SIP, NOT when you press Acquire. Also, leaving a sample on the injection tube with the support arm open will result in loss of sample! Be quick with the support arm when putting tubes in place. Troubleshooting Unblocking a probe Properly prepared samples should not block the machine. Samples should be carefully checked for clumps that can block the probe (filter and/or pipette sample if ANY clumps visible). Check first that the sheath tank is not empty: Remove your sample and Prime several times. If this does not work, run Clean followed by Rinse for 5-10 minutes. Run dh 2 0 for 2-3 minutes before putting your sample back on. Fig Unblocking the sample probe using a syringe. For a persistent clog, it is sometimes necessary to apply a gentle vacuum by syringe - there is a large syringe with rubber tubing in the lab for this purpose. Please ask a member of staff to demonstrate. Make sure that when you prime the machine, there is no tube in place (first, the flow cell empties- flushing out the probe, then the flow cell re-fills). Keeping a tube of Rinse/Clean/dH 2 0 on as you do this is counter-productive, as any debris flushed out will also be taken back up again. *Problematic samples with clumps* Please be aware that if you can see the clump, it is too big! 10

13 De-bubbling filters If the machine has been allowed to run dry you may need to purge air from the filter on the sheath tank and sheath line, and also the flow cell (using Prime). Air can be hard to remove; it is better to prevent this from happening in the first place by checking the tanks! *Knowing how to troubleshoot is important to avoid running into trouble if you will be using the machine after hours* You may be left unable to run the rest of your experiment, or cause subsequent users to be unable to run theirs. This problem may present itself as unexpected dots on your plots (caused by air bubbles, includes both randomly distributed events and low scatter debris-like signals) accompanied by the LSR not showing events from your tube. De-bubbling Filters A B Fig A Purging air from sheath filter: Tap filter gently and check for bubbles. Carefully roll valve and PBS is released along with bubbles. Tilting the tank/filter as you do this in order to encourage the bubbles to move towards the purge valve can also be useful, please ask a member of staff to demonstrate. B Purging air from the sheath line: roll valve (PBS is released, along with bubbles). The cytometer should be on standby while de-bubbling filter/line. 11

14 1.3 Optics Optics Overview Excitation: Light sources: lasers LSRIIA - Blue (488nm), Violet (405nm), UV (355nm), Red (633nm) LSRIIB - Blue (488nm), Violet (405nm), Yellow-Green (561nm), Red (633nm) Fortessa A - Blue (488nm), Violet (405nm), Yellow-Green (561nm), Red (633nm), UV (355nm) Fortessa B - Blue (488nm), Violet (405nm), Yellow-Green (561nm), Red (639nm), UV (355nm) Fortessa C - Blue (488nm), Violet (405nm), Yellow-Green (561nm), Red (639nm), UV (355nm Filters/mirrors: Route the laser beam to the flow cell to interrogate the sample. Collection: Scattered light and any emitted fluorescence are directed by fibre optics to the appropriate block of photomultiplier tubes (PMTs or detectors ) specific for each laser. Filters/mirrors: Used to guide the light to the appropriate PMT in the block. Detectors: Receive the scattered light and fluorescence signals. From these photons, an amplified electrical signal is generated that we observe through the Diva software. Fig Example diagram of LSRII optical bench 12

15 The optical bench of the LSRs see a departure from the layout seen in the FACS Scan, Calibur and Vantage. Instead of passing the light through several mirrors to the correct PMT, the fluorescence from each laser (spatially separated) is directed to the relevant octagonal or triagonal arrangement of PMTs. This results in more efficient collection of the signals. Detection Optics The filters used in the cytometers are optimized for their respective detectors. They can be changed but their transmission characteristics should be known. Gloves should be worn when handling the filters and dichroic mirrors, and they should be held by their edges to avoid leaving fingerprints on the transmission surfaces. The specifications of the filters can be found on the outside edge (by which they should be handled) in small print. A short pass filter (SP), for example 450SP, transmits wavelengths shorter than its specification- i.e. 450nm. Long pass (LP) filters are similarly named, for example 610LP, which transmits light longer than 610nm. Band pass (BP) filters transmit within a given range and are named accordingly. For example, a 710/50BP is a filter that transmits 710nm±25nm (685nm-735nm), 530/30BP transmits nm. Dichroic mirrors can also have a short or long pass function, transmitting longer/shorter than the specification. These are also known as beam-splitters as the rest of the light is reflected. Dichroics are particularly useful for directing light of specific wavelengths down the appropriate route within the optical bench. In front of each PMT is a dichroic long pass (DCLP) mirror. At the first PMT, the longest wavelength to be analysed passes through and everything shorter is reflected to the next PMT. At the second PMT, there is a similar arrangement with DCLP allowing the next longest wavelength to pass and reflecting the rest; and so forth down the PMTs. This set up loses less light than the older collection optics (about 10% is lost passing through the dichroic- generally, mirrors are more efficient at reflecting than filters are at transmitting light). 13

16 Fig Filter processing of light to allow specified wavelengths to be transmitted. The standard configuration of filters is listed inside the hood of the LSRII; it is a good idea to check that the person before you has not changed the filters without returning them to the standard set-up. Please ask a member of the FACS Lab to demonstrate how to change optical filters before attempting to do so yourself. LSRIIA PMT Position LSRIIA Diva Parameter Longpass Filter Blue octagon, A 780/60 blue 735DCLP Blue octagon, B 695/40 blue 680DCLP Blue octagon, C 660/20 blue 635DCLP Blue octagon, D 610/20 blue 595DCLP Blue octagon, E 575/26 blue 550DCLP Blue octagon, F 530/30 blue 505DCLP Blue octagon, G SSC - FSC FSC - Violet trigon, A 530/30 violet 505DCLP Violet trigon, B 450/50 violet - UV trigon, A 525/50 UV 450DCLP UV trigon, B 440/40 UV - Red trigon, A 780/60 red 755DCLP Red trigon, B 730/45 red 685DCLP Red trigon, C 660/20 red - DCLP = Dichroic Long Pass mirror. Fig Standard optical filter configuration in LSRIIA. 14

17 LSRIIB PMT Position LSRIIB Diva Parameter Longpass Filter Blue octagon, A 695/40 blue 685DCLP Blue octagon, B 575/26 blue 550DCLP Blue octagon, C 530/30 blue 505DCLP Blue octagon, D SSC - Blue octagon, E - - Blue octagon, F - - Blue octagon, G - - FSC FSC - Violet trigon, A 660/20 violet 635DCLP Violet trigon, B 605/40 violet 595DCLP Violet trigon, C 560/40 violet 550DCLP Violet trigon, D 510/20 violet 505DCLP Violet trigon, E 450/50 violet - Violet trigon, F - - Y/Green octagon, A 780/60 yellow 735DCLP Y/Green octagon, B 705/70 yellow 690DCLP Y/Green octagon, C 660/20 yellow 635DCLP Y/Green octagon, D 620/40 yellow 600DCLP Y/Green octagon, E 585/15 yellow 570DCLP Y/Green octagon, F - - Red trigon, A 780/60 red 735DCLP Red trigon, B 710/50 red 685DCLP Red trigon, C 660/20 red - DCLP = Dichroic Long Pass mirror. Fig Standard optical filter configuration in LSRIIB. 15

18 Fortessa A PMT Position Fortessa Diva Parameter Longpass Filter Blue octagon, A 780/60 blue 750DCLP Blue octagon, B 695/40 blue 685DCLP Blue octagon, C 610/20 blue 600DCLP Blue octagon, D 580/30 blue 550DCLP Blue octagon, E 530/30 blue 505DCLP Blue octagon, F SSC - FSC FSC - Violet octagon, A 530/30 violet 475DCLP Violet octagon, B 450/50 violet - UV trigon, A 530/30 UV 505DCLP UV trigon, B 450/50 UV - Red trigon, A 780/60 red 750DCLP Red trigon, B 730/45 red 710DCLP Red trigon, C 670/14 red - Y/Green octagon, A 780/60 yellow 750DCLP Y/Green octagon, B 710/50 yellow 685DCLP Y/Green octagon, C 670/30 yellow 635LP Y/Green octagon, D 610/20 yellow 600DCLP Y/Green octagon, E 582/15 yellow - DCLP = Dichroic Long Pass mirror. Fig Standard optical filter configuration in LSRFortessa A. 16

19 Fortessa B and C PMT Position Fortessa Diva Parameter Longpass Filter Blue octagon, A 710/50 blue 685DCLP Blue octagon, B 530/30 blue 505DCLP Blue octagon, F SSC - FSC FSC - Violet octagon, A 670/30 violet 630DCLP Violet octagon, B 610/20 violet 600DCLP Violet octagon, C 586/15 violet 570DCLP Violet octagon, D 540/30 violet 535DCLP Violet octagon, E 525/50 violet 505DCLP Violet octagon, F 450/50 violet - UV trigon, A 450/50 UV 505DCLP UV trigon, B 530/30 UV - Red trigon, A 780/60 red 750DCLP Red trigon, B 730/45 red 690DCLP Red trigon, C 670/14 red - Yellow/Green octagon, A 780/60 yellow 750DCLP Yellow/Green octagon, B 710/50 yellow 685DCLP Yellow/Green octagon, C 670/30 yellow 635LP Yellow/Green octagon, D 610/20 yellow 600DCLP Yellow/Green octagon, E 586/15 yellow - DCLP = Dichroic Long Pass mirror. Fig Standard optical filter configuration in LSRFortessa B and C. 17

20 LSRII (A) Blue Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

21 LSRII (A) Trigons: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

22 LSRII (B) Blue Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

23 LSRII (B) Yellow-Green Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

24 LSRII (B) Violet Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

25 LSRII (B) Red Trigon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

26 LSRFortessa A Blue Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

27 LSRFortessa A Yellow/Green Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

28 LSRFortessa A Violet Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

29 LSRFortessa A Trigons: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

30 LSRFortessa B and C Blue Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

31 LSRFortessa B and C Yellow/Green Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

32 RFortessa B and C Violet Laser Octagon: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

33 LSRFortessa B and CTrigons: filter optics and collection pathway BP = Band Pass filter DCLP = Dichroic Long Pass mirror Figure

34 1.4 Digital Electronics Digital flow cytometry and the LSRs The LSRs are BD s top of the range benchtop analyser, offering flexibility to multi-colour applications. They are faster: the electronics are capable of digitising signals 10 million times per second; there is increased processing capability due to reduced electronic dead time. They have better resolution: improved ADC (increased bit size data) and electronics giving better channel resolution and detection sensitivity. We can have a full compensation matrix: inter-laser compensation is possible and can be applied or changed on/offline. More information can be obtained and digital pulse processing of all parameters is possible. Digital pulse processing In basic terms, analog signals have a continuous distribution (i.e. they can take any value within a defined range) and digital signals are a discrete distribution (i.e. can only have certain binned values the number of which is determined by the digitisation). Ultimately, all cytometers are digital but they differ in where digitisation takes place. In any case the more discrete channels that a distribution can be divided into, the better the stored data relates to original data. Analog: PMT Compensation LIN/LOG Transformation Digitise (by ADC) Digital: PMT Digitise (by ADC) Compensation LIN/LOG Transformation Fig Order of data processing in analog vs. digital systems. In the LSRs, signals from the detector (PMT) are continuously digitised by an Analog to Digital Converter (ADC). The ADC captures a snapshot of the electric voltage from the PMT and represents it as a digital number that can be sent to a computer. By capturing the voltage millions of times per second (millions of time slices ), you can get a very good approximation to the original light signal. The ADC in the LSRs for height is 14-bit, this gives i.e. 16,384 channels available (16, 384 levels that the signal may be assigned). Each measurement represents the height of the pulse at that time. The area ADC gives 18-bit data i.e or 262,144 channels. If we add together all the time slices of a pulse, we get the area, i.e. the total amount of fluorescence coming from the cell. 32

35 If we attach an oscilloscope to a detector on a flow cytometer, we can see the voltage pulse that is generated: Photon of light Photomultiplier Tube (PMT) Voltage pulse Fig Photon conversion by PMT to a voltage pulse. We can measure the height of the total pulse by looking at the output of the ADC (channel number read-out): Pulse Height 16,384 Photon of light Photomultiplier Tube (PMT) 0 Fig PMT generated pulse: pulse height readout from ADC. The area of the pulse is determined by adding the height values for each time slice of the pulse (which is determined by the speed of the ADC, which is 10MHz i.e. 10 million per second or 10 per microsecond). The area is a better representation of the total amount of fluorescence: Photon of light Photomultiplier Tube (PMT) Pulse Area (between 0-262,144) Fig PMT generated pulse: pulse integral readout from ADC. 33

36 The width parameter can be thought of as being directly related to the time that a particle of interest takes to flow past through the laser beam. Fig Time taken to pass through laser is proportional to width parameter. However, in the LSR, this is a calculated rather than a measured parameter. Because the width can also be taken as the number of time slices sampled, this is calculated as Area/Height x 64K. Because fluorescence signals from the PMTs are digitised immediately in digital cytometers, there is the advantage that noise introduced by other electronic circuitry is reduced, and cytometer dead time is reduced. The signals are sampled over tiny time slices and each given a value; digitised values are retained in buffer memory until the area integral and pulse width measurements are completed. The digital values are then passed to other electronics such as gating boards, before interfacing with the computer workstation (Diva software). Note: the data is stored as linear, if you wanted to perform log calculations, the computer looks up the correct value using a Look Up Table. The fcs 3 files contain the uncompensated values, (but the compensation matrix is also saved) meaning that compensation can be augmented post collection (remember that compensation on saved analog files cannot be taken off - it is possible to add compensation using analysis software but too much compensation is unsalvageable). The width is a processed parameter on the LSR; H and A are both measured but we generally use the A parameter on LSR because it represents total fluorescence coming from the cell. 34

37 2.1 Commonly Used Fluorochromes Laser 488nm Blue 633nm Red 407nm Violet 355nm UV Collection Filter (bandpass) Diva Parameter Name 530/30 530/30 blue 575/26 575/26 blue Fluorochromes FITC, Alexa 488, egfp, CFSE, Dye Cycle Green PE, Cy3, Sytox-Orange, Dye Cycle Orange 610/20 610/20 blue PI, PE-Texas Red, PE-Alexa Fluor /20 660/20 blue 695/40 695/40 blue 780/60 780/60 blue PE-Cy7 660/20 660/20 red PE-Cy5 (also Tricolour or Cychrome ), 7AAD PerCP, PerCP-Cy5.5, DRAQ5, Dye Cycle Ruby APC, Alexa 633, Alexa 647, TO-PRO- 3, Cy5, DRAQ5, DRAQ7, Dye Cycle Ruby 730/45 730/45 red APC-Cy5.5, DRAQ7, Dye Cycle Ruby 780/60 780/60 red APC-Cy7, DRAQ7, Alexa Fluor /50 450/50 violet Alexa 405, CFP, Pacific Blue, DAPI, Hoechst (blue), Celltrace Violet, Dye Cycle Violet, BV421, VPD450, Calcein Violet 450, Fixable Viability Dye efluor 450, Cell Proliferation Dye efluor /30 530/30 violet Qdot 525, Pacific Orange, Krome Orange 440/40 440/40 UV Celltrace Violet, DAPI, Hoechst (blue) 525/50 525/50 UV PI Fig This table gives examples of the fluorochromes most commonly used on LSRIIA, showing which laser they are excited by and which filter should be used to collect the signal. All of these fluorochromes can be used with the standard LSRII A filter arrangement as shown in section

38 Laser 488nm Blue 633nm Red 407nm Violet 561nm Yellow- Green Collection Filter (bandpass) Diva Parameter Name 530/30 530/30 blue 575/26 575/26 blue 695/40 695/40 blue 660/20 660/20 red 710/50 710/50 red Fluorochromes FITC, Alexa 488, egfp, CFSE, Dye Cycle Green PI, Cy3, Sytox-Orange, Dye Cycle Orange PerCP, PerCP-Cy5.5, PerCP-eFluor710, DRAQ5, Dye Cycle Ruby APC, Alexa 633, Alexa 647, TO-PRO-3, Cy5, DRAQ5, DRAQ7, Dye Cycle Ruby APC-Cy5.5, Alexa 700, DRAQ7, Dye Cycle Ruby 780/60 780/60 red APC-Cy7, DRAQ7, Alexa Fluor /50 450/50 violet Alexa 405, CFP, Pacific Blue, DAPI, Hoechst (blue), Celltrace Violet, Dye Cycle Violet, BV421, VPD450, Calcein Violet 450, Fixable Viability Dye efluor 450, Cell Proliferation Dye efluor /20 510/20 violet Qdot 525, BV510, BD Horizon V500, 560/40 560/40 violet Qdot 565, Pacific Orange, Krome Orange, BV /40 605/40 violet Qdot 585, Qdot 605, BV /40 660/40 violet Qdot 655, BV /15 585/15 yellow PE, Alexa Fluor /40 620/40 yellow PE-Texas Red, M-Cherry, M-RFPMars 660/20 660/20 yellow PE-Cy5 (also Tricolour or Cychrome ), 7AAD, E2-Crimson, M-Plum 705/70 705/70 yellow PE-Cy5.5, DRAQ7 780/60 780/60 yellow PE-Cy7, DRAQ7 Fig This table gives examples of the fluorochromes most commonly used on LSRIIB, showing which laser they are excited by and which filter should be used to collect the signal. All of these fluorochromes can be used with the standard LSRIIB filter arrangement as shown in section

39 Laser 488nm Blue 561nm Yellow- Green 355nm UV 633nm Red 405nm Violet Collection Filter (bandpass) Diva Parameter Name Fluorochromes 530/30 530/30 blue FITC, Alexa 488, egfp, CFSE, Dye Cycle Green 580/30 575/26 blue PE, Cy3, Sytox-Orange, Dye Cycle Orange 610/20 610/20 blue PI, PE-Texas Red 695/40 695/40 blue PerCP, PerCP-Cy5.5, PerCP-eFluor710, DRAQ5, Dye Cycle Ruby 780/60 780/60 blue PE-Cy7 582/15 582/15 yellow PE, Alexa Fluor /20 610/20 yellow PI, PE-Texas Red, M-Cherry, M-RFPMars 670/30 670/30 yellow PE-Cy5 (also tricolour or cychrome ), 7AAD, E2-Crimson, M-Plum 710/50 710/50 yellow PE-Cy5.5, DRAQ7 780/60 780/60 yellow PE-Cy7, DRAQ7 450/50 450/50 UV DAPI, Hoechst (blue), Celltrace Violet 530/30 530/30 UV PI 670/14 670/14 red APC, Alexa 633, Alexa 647, TO-PRO-3, Cy5, DRAQ5, DRAQ7, Dye Cycle Ruby 730/45 730/45 red APC-Cy5.5, DRAQ7, Dye Cycle Ruby 780/60 780/60 red APC-Cy7, Alexa Fluor 750, DRAQ7 Alexa 405, CFP, Pacific Blue, DAPI, Hoechst (blue), Celltrace Violet, Dye 450/50 450/50 violet Cycle Violet, BV421, VPD450, Calcein Violet 450, Fixable Viability Dye efluor 450, Cell Proliferation Dye efluor 450, VioBlue Qdot 525, BV510, BD Horizon V500, 525/50 525/50 violet Fixable Viability Dye efluor 506, VioGreen Fig This table gives examples of the fluorochromes most commonly used on LSRFortessa A, showing which laser they are excited by and which filter should be used to collect the signal. All of these fluorochromes can be used with the standard LSRFortessa A filter arrangement as shown in section

40 Laser 488nm Blue 561nm Yellow- Green 355nm UV 639nm Red 405nm Violet Collection Filter (bandpass) Diva Parameter Name 530/30 530/30 blue 710/50 710/50 blue 586/15 586/15 yellow PE, Alexa Fluor 555 Fluorochromes FITC, Alexa 488, egfp, CFSE, Dye Cycle Green PerCP, PerCP-Cy5.5, PerCP-eFluor710, DRAQ5, Dye Cycle Ruby 610/20 610/20 yellow PI, PE-Texas Red, M-Cherry, M-RFPMars 670/30 670/30 yellow PE-Cy5 (also Tricolour or Cychrome ), 7AAD, E2-Crimson, M-Plum 710/50 710/50 yellow PE-Cy5.5, DRAQ7 780/60 780/60 yellow PE-Cy7, DRAQ7 450/50 450/50 UV DAPI, Hoechst (blue), Celltrace Violet 530/30 530/30 UV PI 670/14 670/14 red APC, Alexa 633, Alexa 647, TO-PRO-3, Cy5, DRAQ5, DRAQ7, Dye Cycle Ruby 730/45 730/45 red APC-Cy5.5, DRAQ7, Dye Cycle Ruby, 780/60 780/60 red APC-Cy7, Alexa Fluor 750, DRAQ7, Fixable Viability Dye efluor 780 Alexa 405, CFP, Pacific Blue, DAPI, Hoechst (blue), Celltrace Violet, Dye 450/50 450/50 violet Cycle Violet, BV421, VPD450, Calcein Violet 450, Fixable Viability Dye efluor 450, Cell Proliferation Dye efluor 450, VioBlue Qdot 525, BV510, BD Horizon V500, 525/50 525/50 violet Fixable Viability Dye efluor 506, VioGreen 540/30 540/30 violet 586/15 586/15 violet BV /20 610/20 violet BV /30 670/30 violet BV650 Fig This table gives examples of the fluorochromes most commonly used on LSRFortessa B and C, showing which laser they are excited by and which filter should be used to collect the signal. All of these fluorochromes can be used with the standard LSRFortessa B filter arrangement as shown in section

41 *Which LSR? * The configurations of the four LSRs are different (see section 1.3); this may mean that one machine is more appropriate than the other for a specific experiment. These applications can only be run on a specific LSR: -LSRIIA or LSRFortessas: Hoechst side population and Indo-1 calcium flux. -LSRIIB or LSRFortessas: RFP and variants. These applications may be better using the yellow laser on LSRB or LSRFortessas: -Any experiment using PE and PE tandems (there is less autofluorescence and better S/N ratio). -Experiments combining GFP or CFSE with PE. Note: For PerCP or PerCP variants (PerCP-Cy5.5 for example), use 488 (blue) laser excitation NOT the 561nm (yellow). 2.2 Fluorochrome Selection There are many factors that will affect the choice of fluorochromes for an experiment. Please speak to a member of the FACS Lab when designing a new experiment and before purchasing expensive reagents! Viability dyes The presence of dead and dying cells in a sample can have a detrimental effect on the analysis and interpretation of fluorescence data. Dead cells may lose markers present on/in live cells and can become autofluorescent, i.e. giving a positive fluorescent signal although no fluorochrome is present. They may also non-specifically take up antibodies, generating artefacts that are difficult to exclude when you wish to analyse your data. These problems are easily overcome by using a viability dye, sometimes referred to as a live/dead dye. These dyes are excluded by the intact membranes of live cells, but can enter dead and dying cells. Therefore, gating on the population negative for the viability dye easily excludes dead cells. 39

42 Dye Excited by: Collected in (LSRIIA/B - LSRFortessa A/B): Propidium iodide (PI) Blue Laser/Yellow laser 610/20bp / 575/26bp 7AAD Blue Laser /Yellow laser 660/20bp / 610/20bp TO-PRO-3 Red Laser 660/20bp DAPI UV laser / Violet laser 440/40bp / 450/40bp / 450/50bp DRAQ7 Red laser 670/14bp / 660/20bp / 710/50 bp / 730/45bp /780/60bp Fig. 2.2: Examples of commonly used viability dyes. *Tips and Tricks* The dyes mentioned above can only be used in unfixed samples. In fixed samples the dye would enter all cells. The most popular viability dye for the LSRs is DAPI. This is because it is excited by the violet and UV lasers and rarely overlaps with another fluorochrome in the same experiment. Fixable LIVE/DEAD amine reactive dyes If you wish to be able to fix your sample, you may try using the amine-reactive dyes from Molecular Probes, Invitrogen. In membrane-compromised cells, the dye has access to free amines both in the cell interior and on the cell surface; in viable cells, the dye can react only with cell-surface amines, generating lower levels of fluorescence. Up to 50- fold difference in intensity can be obtained between live and dead cells, and the staining is preserved following formaldehyde fixation. Availability This may be the major factor limiting your choice of fluorochrome combination. Although a selection of different directly conjugated fluorochromes are available for commonly used antibodies such as CD4, this may not be the case with less common antibodies. In many cases it is necessary to use a two-step staining process involving primary and secondary antibodies and in this case great care must be taken to avoid non-specific staining and cross-reaction between antibodies. Please ask a member of the FACS Lab if you need further advice on this. 40

43 Physical Characteristics Physical characteristics of fluorochromes may influence their selection, for example, the size of different fluorochromes varies considerably and when doing intracellular staining it may be necessary to select a smaller fluorochrome. The brightness (quantum efficiency) of different fluorochromes also varies. For multi-colour staining it is advisable to use the brightest fluorochromes to identify the dimmest or least expressed markers. 2.3 Combining fluorochromes Spectral overlap With all flow cytometers, problems can be encountered when using multiple fluorochromes because the emission of each fluorochrome can spill over into the detectors of others. This problem increases with the number of fluorochromes used and is therefore especially relevant to the LSRs. Fig Emission spectrum of two commonly used fluorochromes. FITC (green line) is detected with a 530/30 filter and PE (purple line) is detected with a 575/26 filter. These filters are chosen to capture the maximum emission of their respective fluorochromes. However, some FITC emission can be seen spilling over into the 575/26 spectral region and dim PE emission can be seen in the 530/30 and 660/20 (PE-Cy5) regions. Problem pairs Because of the spectral overlap, certain combinations of fluorochromes can be problematic, in many cases this will depend on how bright the fluorochromes are. For example, combining a very bright GFP with dim PE could be difficult, but a dimmer GFP 41

44 signal may not cause problems. Other combinations will always be potentially difficult, an example of this is given in Fig below. Fig Spectral overlap of PE-Cy5 (excitation dark green line, emission light green line) and APC (excitation dark purple line, emission light purple line). PE-Cy5 is optimally excited by the 488 laser and APC by the red laser, however some excitation of the Cy5 component of PE-Cy5 can occur with the red laser. Both fluorochromes emit in the 660/20nm region so it can be very difficult to separate the red excited PE-Cy5 signal from the APC; take care in checking the voltages for APC and PE-Cy5 to obtain a good balance of signals and still be able to compensate PE-Cy5 out of the APC channel. Example combinations Fig colour staining, using FITC (blue line) and APC (purple line). The emission of these two fluorochromes is well separated resulting in very little spectral overlap. If using live (unfixed) cells DAPI should be added to this combination as a viability dye. 42

45 Fig colour staining, using FITC, APC and PE-Cy7 (yellow line). The emission of PE-Cy7 is higher than that of both FITC and APC so there is little spectral overlap. If using live (unfixed) cells DAPI should be added to this combination as a viability dye. *Useful websites: * The following websites all enable you to view the excitation and emission of various fluorochromes and compare different fluorochromes to determine the likelihood and severity of spectral overlap

46 2.4 Compensation Compensation is used to control the effects of spectral overlap. The aim of compensation is to account for spectral overlap of fluorochromes into adjacent detectors. This is very important for the reasons explained in section 2.3 above. The LSR has a number of features that help to improve the ease and accuracy of compensation. Having the correct controls is also very important. For further explanation on the principles of Compensation, please speak to any member of the FACS Lab. Controls For each of the fluorochromes used in the test samples, a single colour positive control is needed for compensation. Each control should ideally be made up of a population of negative or unstained cells and a population of single colour positive cells. Both the positive and negative cells should have the same level of background autofluorescence, ideally this will mean ensuring that they are the same type of cell, e.g. lymphocytes. Ideally the positive and negative populations in the controls should both be large, well defined and well separated. However, if you are looking for a rare population or have a very limited number of cells this might not be possible. There are three ways to overcome this problem: 1. Use different cells for your compensation controls - e.g. if the test samples are primary tissue in limited supply, find a cell line that expresses the markers of interest and use these for the compensation. 2. Use a different antibody conjugated to the same fluorochrome if a marker of interest is rare or possibly absent in the control cells, it is better to use a different antibody, directed against a more common marker, but carrying the same fluorochrome as will be used in the test. CD45 antibodies are often used for this purpose when studying rare lymphocyte markers. It is important to note however that the exact same fluorochrome should be used, e.g. GFP and FITC may both emit a signal detected in the 530/30 channel, but they are not the same fluorochrome. Tandems are trickier due to variations in chemical conjugation: if a tandem conjugate is being used, e.g. PE-Cy5, the same batch of tandem should be used. 44

47 However, it is safer still to use a cell line highly expressing the marker and label with the same antibody you are planning to use. 3. Use compensation beads: If the marker of interest is rare or absent on/in the control cells, you can still use the antibody if you substitute the cells for comp beads. Comp beads are polystyrene microparticles, available as Anti-Rat or Antimouse Ig, kappa. They bind to rat or mouse light chain Ig antibodies (check which ones are right for your antibodies). Beads with no binding capacity are also available and can be used as a negative control to determine background fluorescence, ensuring that clear positive and negative populations are present in all compensation controls. Compensation Beads To prepare the controls the antibodies to be used are incubated with the beads as follows: Combine 100µl staining buffer + one drop negative beads + one drop of Comp beads (Anti-mouse or Anti-rat) + antibody (at the concentration used in the experiment). Incubate for 30min (RT in the dark). Wash with 2ml of staining buffer and resuspend compensation controls in 500µl staining buffer and run them on the flow cytometer. Cells can also be added to the compensation control tubes to check for autofluorescence from your cells and to help establish your baseline voltages. A B Fig A Scatter plot showing a large scatter gate that includes the Comp beads and cells. B APC-Alexa750+ve beads can clearly be distinguished from unstained particles and used for compensation (plot showing only scatter gated events). 45

48 The Bi-exponential Display The bi-exponential display is a feature of digital cytometers. It allows you to see below 0 on a log axis. This means that any events that would normally appear crushed on the axis will be visible; the bi-exponential display does not change the data, it merely transforms it to present it in a visually understandable way. This makes it much easier to do compensation, and is particularly useful for seeing if a sample has been over compensated, as shown below. A B C D E Fig A shows an uncompensated sample on the standard LSR 5 log display, B shows the same uncompensated sample viewed using the bi-exponential display. C shows the sample once compensation has been applied by eye without using the biexponential display, the centre of the positive population appears to be roughly in line with the centre of the negative population. D shows what the compensation used in C looks like when viewed with the bi-exponential display. It can clearly be seen that the sample is actually over compensated, with the positive events below the negatives. E shows correct compensation, as viewed on the bi-exponential display. 46

49 The bi-exponential display function can be selected for individual axes and plots. To switch it on: select the plot, then in the Instrument window check the boxes for the relevant axes. Note: The scale of the axis will automatically adjust to give the best display of all current events. During sample acquisition this can lead to large blank areas appearing in the negative region, however this should be corrected when all the collected data is displayed for analysis. You may find it best to turn off the bi-exponential display when acquiring data. Compensation median fluorescence technique It is recommended that you use the statistics functions in Diva to calculate the exact amount of compensation required. This is done by drawing gates around the positive and negative populations and comparing the median fluorescence of the two. For example, if you are compensating FITC out of PE, the median fluorescence of the double negative population will be X. The FITC + PE - cells have no PE and should therefore give the same median PE signal as the double negative (X). However, because of the spectral overlap of the FITC signal into the PE channel, the FITC + PE - population has an increased median PE fluorescence of Y. To correctly compensate this sample you simply increase the amount of compensation applied until Y=X. You just have to imagine a line in the centre of the positive population that needs to be lined up with the centre of the negative population. You will be taken through the process of compensation in section 3. *Tips and Tricks* If you are using any fluorochrome pairs that could be difficult to compensate, e.g. PE- Cy5 with APC, it is a good idea to run these first and do a rough check on the compensation by eye before saving all the samples. This prevents you from having to re-record all your controls if you need to change the voltages on one to help compensation. After checking the compensation reset it to zero before recording the sample. 47

50 Copying the spectral overlap Once you have calculated your compensation you will need to apply it to all future tubes. This can be done by just writing down the compensation applied to each tube and typing it in for the sample tube, or by using the copy spectral overlap function. For the latter, once you have set the compensation for a control, expand the tube and right click on instrument settings. Select Copy spectral overlap then right click on the instrument settings for the experiment and select Paste spectral overlap. Repeat this procedure after compensating each control. Having completed the compensation, click Next; the new tube and all subsequent tubes should contain all the compensation and be ready for you to run your samples. There is an example experiment in section 3 where you can try this out. Automatic compensation As the number of fluorescence parameters in an experiment increases, compensation becomes increasingly difficult to set manually. For a six-colour experiment, 30 spectral overlap values need to be adjusted, and for an eight-colour experiment, 56 values need to be adjusted. The process of manually correcting spectral overlap values can take several hours and is very difficult to set accurately. The Compensation Setup feature in BD FACSDiva software is designed to automatically calculate spectral overlap values for an experiment, saving time and eliminating the inaccuracies introduced with manual compensation. We recommend that for any experiment with three or more fluorochromes compensation should be set using the automatic feature. Compensation Setup is designed to work with single-stained controls. These controls can consist of single-stained cells or capture beads. An unstained control is required as well, in a separate tube or in the same tube as the single-stained controls. As mentioned, you can find more details about the process of compensation in section 3. 48

51 We will now run an example experiment with cells, FITC and PE beads. Open the software (double click the FACSDiva icon on the desk top). There is no password, just hit Enter at the password window. *Checklist before starting experiment* Has the LSR been switched on long enough for the lasers to warm up? Is the sheath tank full and waste tank empty? Is the standard configuration of optical filters in place? Is it clean (who used it last? when was it used last?) - Worth giving a few Primes, checking cleanliness with dh The Browser, Worksheet, Inspector and Acquisition windows 1. Browser 2. Cytometer 5. Worksheet 4. Acquisition Dashboard 3. Inspector Fig Diva v6 software windows 50

52 1. The Browser: where you can make new folders and experiments, and access or delete acquired experimental data. Fig The Browser window 2. Cytometer: controls all instrument related electronic functions. This window cannot be accessed unless an experiment is open, and the arrow to the left of a tube in the browser is selected (green). Instrument connection status is shown in the footer of the window. There are a number of tabs below the header including Parameters (equivalent to Detectors and Amps in CellQuest) and Compensation; these will be discussed further in later sections. 3. Inspector: Each Diva window has information that can be edited/changed via the Inspector, which is accessed by making a window active. 4. Acquisition Dashboard: is the main panel for collecting data. Here you can start acquiring/recording data, and add storage and stopping gates for acquisition. Changes to the number of events displayed and recorded can also be made here. Data recording progress is shown as a coloured bar; when completed, click the NEXT TUBE icon to move on to the next sample. The sample flow rate (events/sec), aborted events and time elapsed is also shown. 5. The Worksheet: where you can make plots and histograms. There are two types of worksheets but we only use the Global type worksheet to acquire data. Data analysis is performed using FlowJo. 51