Independent End User Training. Curriculum and Supporting Materials. DHVI BD LSRII (L01/L02) and BD LSRFortessa (F01)

Transcription

1 Shared Resources: Flow Cytometry and Cell Sorting Independent End User Training Curriculum and Supporting Materials DHVI BD LSRII (L01/L02) and BD LSRFortessa (F01) Revised 1/2016, PMM, GDS

2 1.0 Purpose / Scope The purpose of this training packet is to provide materials to support and enhance your hands-on training experience. 2.0 Expectations Trainees must have been recommended by their manager/mentor to undergo training, have at least 10 instrument contact hours with an experienced flow cytometer user, have a need to use DHVI Flow Facility instruments at least 5 hours/month and have a strong desire to learn how to safely and correctly operate a flow cytometer to acquire comprehensive polychromatic flow data. Trainees must complete two BD Biosciences online courses, Introduction to Flow Cytometry and BD FACSDiva Software v8 Overview, PRIOR to initiating hands-on training. Trainees to will be expected to complete all three components of the DHVI Flow Facility Training program: Classroom group orientation (~2hr), Hands on session (~2 hr) and a Practical Exam (~2 hr). If the trainee misses one of the sessions or fails the practical exam, they will not be certified by the assigned training Operator and will have to repeat the training program at a later date (see attachment #2). The Pin Code to access Bay 5 will be given to the Trainee at the classroom session. If the Independent End User is the last person to leave the room they need to make sure that the door(s) are closed and locked. All approved Independent End Users of the DHVI Flow Facility instruments are expected to fully comply with the Policies and Procedures for DHVI Flow Facility Users detailed in SOP Flow_010 (on website) that is covered in the classroom session of the training. 3.0 Cytometer Overview Your trainer will orient you to the critical components of the cytometer: Fluidics, Optics, Computers, and the BDFACS Diva software (see attachments #3-5). 4.0 Operation and Maintenance 4.1 Instrument Startup Turn on Fluidics Cart (L02 only) Turn on Cytometer (needs 30 minute laser warm up) Be sure Sheath tank is full: Filling L02 Sheath Tank: Unscrew large cap and carefully pour sheath into the tank up to the top line on the tank Filling L01/F01 Sheath Tank: Depressurize the sheath tank by slowly pulling up on the pressure relief valve Remove the lid of the sheath tank, and fill up to the top weld line inside the tank. Revised 1/2016 Page 2 of 26

3 DO NOT OVER FILL Replace the lid making sure that you lock it down from side to side, not top to bottom Making up Sheath Fluid (PBS): x sheath containers are located under the sink between Bay 5 and Bay 6. Fill the sheath container with dh 2 O to the Fill Line on the back of the container Put a large X on the cap and the sides of the sheath container to indicate that it has been diluted. DO NOT X THE LABEL If you spill the sheath while pouring, please clean it up Additional sheath is located under the desk behind the L Empty Waste tank if full: Emptying Waste Tank: There are two (2) waste tanks used with the L01/L02/F01 because the waste needs to sit in the bleach solution for at least 24 hours before emptying down the sink L02: Bring the spare waste tank to the sink. Remove the large cap. Turn on the cold water and pour the 10% bleach waste down the drain letting the water run for a couple minutes when finished to flush the pipes Put 1 L of bleach and 2 L of water into the waste tank, so that when the tank is full it will be a 10% final bleach solution The bleach is located under the sink between Bay 5 and Bay Additional bleach is located under the desk behind the L Remove the RED filter waste cap from the full waste tank and put it on the empty waste tank going onto the fluidics cart. Unscrew the sensor and carefully pull it out of the tank and put it in the empty waste tank. Be careful not to break the filter on the tubing NOTE: The red filter waste cap ALWAYS goes on the waste tank on the fluidics cart!! Put the large white cap and the small cap on the full waste tank and put the empty after date on the tape. Put the waste tank in the bucket to the side of the fluidics cart. Revised 1/2016 Page 3 of 26

4 L01/F01: Disconnect the orange waste line and the black waste sensor line from the white cap on the full waste tank Bring the spare waste tank to the sink. Remove the large cap. Turn on the cold water and pour the 10% bleach waste down the drain letting the water run for a couple minutes when finished to flush the pipes Remove the waste tank from the holder. Put the empty waste tank in the holder Put the white waste cap with the sensor connections on it on the empty waste tank Reconnect the waste and sensor lines Put the large white cap on the full waste tank and put the empty after date on the tape Put the full waste tank in the bucket to the side under the L01/F01 table L02 Only. Be sure the Sheath Reservoir has ~1/4 of sheath in it. See Attachment #8 for troubleshooting the sheath reservoir Filling Sheath Reservoir: Put fluidics in Standby Unscrew the white cap and take sensor out of the tank Using the funnel (located at the sink), put ~1/4 of sheath fluid in the tank and replace the sensor Low Sheath/Sheath Alarm Put the cytometer in standby and refill the sheath tank After you refill the tank, you need to restart the fluidics cart NOTE: If you do not restart the cart, the sensor doesn t sense the sheath and will drain the reservoir causing what can be a lengthy troubleshooting procedure that you will be billed for at Operator rates Check Sheath Filter for air bubbles Hold the sheath filter so that the tubing connected to the stopcock/vent cap is facing you Tap the filter a couple of times to dislodge any bubbles that might be in there Remove air bubbles from sheath filter: L02: Using the stopcock device. Revised 1/2016 Page 4 of 26

5 Using a container to catch the sheath that comes out, roll back the stopcock to un-pinch the tubing Discard sheath into container until all bubbles are gone, and then roll back the stopcock to pinch the tubing to stop the flow of sheath L01/F01: Using the vent port on the top of the filter Using a container to catch the sheath that comes out, pinch off the tubing right below the vent cap, and remove the cap Discard sheath into container until all bubbles are gone, and then replace the vent cap so that it pinches off the tubing to stop the flow of sheath Priming Cytometer to remove Air Bubbles NOTE: This has to be done every time the cytometer is turned on because of the flow cell emptying and refilling with sheath Remove the tube of dh 2 O from SIP and press prime times or more if necessary to remove any air inside the flow cell Note: Never prime the cytometer with a tube on the SIP to prevent a potential clog going into the flow cell. 4.2 Instrument Shutdown If you are the last User of the day, you need to shut down the cytometer, fluidics cart (L02) and log off the computers Bleach: Run 5 minutes of 10% Bleach on HI dh 2 0: Run 5 minutes of dh 2 0 on HI. You need to run water at least as long as you run bleach, so that you get all the bleach out of the tubing to prevent corrosion. Revised 1/2016 Page 5 of 26

6 4.2.3 Standby: Set the fluidics control to Standby and Low and leave a tube of NO MORE THAN 1 ml of dh 2 0 on the SIP The tube of water is to keep the sheath from crystallizing in the injection probe You CANNOT have more than 1 ml in the tube because when the cytometer is turned off, or left in standby mode, there is a small amount of fluid that back drips into the tube. Too much fluid in the tube could over flow into the air line causing a LENGTHY service call to be placed Shut Off the Fluidics Cart (L02) and Cytometer as well as logging off of the computers NOTE: Please DO NOT shutdown the computers, we need access Top Off the Sheath tank (see above) Empty Waste if necessary (see above). 5.0 Cytometer Setup and Tracking (CS&T) CS&T needs to be run EVERY time the cytometer is turned on, to ensure consistent laser performance from day to day. Lasers are light bulbs, with use they can blow out at any time. 5.1 Log into the Cytometer Controller computer => launch the FACSDiva software => click OK in the Administrator window: L01: Username: userl01; Password: Msrb L02: Username: userl02; Password: Msrb F01: Username: userf01; Password: Msrb If you start the software before the cytometer, you will need to manually connect them by selecting the Instrument menu => Connect. 5.3 The instrument Status will probably say Fluidics Not Ready, just click clear. 5.4 Making CS&T Beads: Beads are located in the refrigerator behind the L01. Vortex the beads and place 3-4 drops of beads/500 ul of PBS. Please make sure that you are using the correct CS&T bead lot number which can be found in the CS&T software Label tube with: CS&T, lot # (found on CS&T vial), date and your three initials. 5.5 Running CS&T Beads: Select Cytometer in Workspace menu => CS&T. Revised 1/2016 Page 6 of 26

7 5.5.2 The cytometer is now disconnecting from the Diva software and connecting to the CS&T software Check the Load Tube Manually box (if not already checked) Make sure the CS&T Lot ID matches the CS&T Beads being used Put the Cytometer in Low and Run, Vortex and Load the CS&T beads Click Run in the CS&T software, click OK. The CS&T will take about 8 minutes to complete When completed, remove the CS&T beads and put back into the refrigerator If CS&T: Passed: Exit CS&T Program => after cytometer connects to the Diva software, proceed as usual Passed with Warnings (PWW): Same as step , but notify the Flow Facility about the warning Failed: Try making fresh beads, double check Lot ID and /or shutdown and restart the computer and cytometer and repeat process. If still failed, please contact the Flow Facility before continuing If no Flow Facility personnel are available: Exit the CS&T software and run the CS&T beads in the manual CS&T experiment located in the Diva Browser Browser => Manual CS&T folder => Double click CS&T experiment to open it => Expand the specimen => Copy and paste the prior tube and rename it with the correct date and lot number Run the CS&T beads to make sure that the dim and bright bead populations show up in the FCS/SSC plot (indicates that the blue laser and FCS/SSC PMTs are working) and that all three fluorescent peaks show up in their relative histograms (indicates all lasers/pmts are working). If all look good, record 1,000 events and continue on as usual with your experiment NOTE: It is important that you report any warnings/failures to the Flow Facility so that we can monitor the lasers to prevent a blowout that could shut down the cytometer for use for a lengthy period of time NOTE: To see if CS&T has been performed for that day: Click on the CS&T Reports folder on the desktop => Config folder => Year Folder => Month Folder => Day Folder =>.pdf of the CS&T Report. Revised 1/2016 Page 7 of 26

8 6.0 Data Collection NOTE: The Reset Target Values and Define Baseline procedures will always be done by the Flow Facility. If you get a notice saying that the Baseline has expired, please contact the Flow Facility. 6.1 Before Running Samples: Log onto the Workstation computer (posted instructions on Quick Fact sheet hanging on the wall next to the computer) Open Internet Explorer Open Fac Man => Log in using your DHE => Start your Fac Man session Open Biotrue Uploader and log in (posted instructions on Quick Fact sheet hanging on the wall next to the computer). Click continue through the security windows if applicable) Open the BDExort folder => FCS folder => Day s folder. 6.2 Creating a New Experiment in Diva Browser: Make a Yearly, Monthly and/or Daily folder if necessary and name accordingly in the Browser Frame => right click on Administrator => add a New Folder for the Year and name it accordingly => right click on the Yearly folder => add a folder for the Month and name it accordingly => right click on the Monthly folder => add a folder the Day and name it accordingly Right click the Daily Folder => New Experiment => 10 Color Template (L01/L02) or 8 Color Template (F01) => OK The Experiment should default open, but if it does not, double click on the Experiment Book to open it Right click on the Experiment and rename it with the date (yymmdd), run number, machine number, your initials and experiment number (e.g L02 PMM005) Right click on the Specimen and rename it with the date, your initials and experiment number (e.g PMM005) Expand the Specimen and right click on the Tube_001 and rename it with your initials_001 (e.g. PMM_001) Click the Acquisition Tube Pointer Box to the left of the tube in the Browser Frame => click the Parameters Tab in the Cytometer Frame => delete any unwanted parameters by holding down the control key => select the dot next to the parameter you want to delete => click on delete (this saves on memory) NOTE: We do suggest collecting one empty parameter for gating out autofluorescence if needed. Revised 1/2016 Page 8 of 26

9 6.3 Creating Global Worksheets: A new experiment (created from the icon and not the menu) will default with an empty Normal Worksheet. Normal Worksheets are tube specific, so we recommend using a Global Worksheet which is experiment specific To create a Global Worksheet, right click the Experiment book => New Global Worksheet Click on the worksheet to add plots Make the number of plots needed to satisfy your experimental design/panel, and change the labels accordingly You will need at least one dot plot of FSC/SSC, one dot plot with two parameters for compensation and a histogram for each parameter you are using (including the dump parameter if you are using one) to set your voltages Click on the Dot plot icon in the Worksheet toolbar, and then click on the worksheet Click on the parameter description to change the parameter labels. (i.e. FSC/SSC) NOTE: If you have FSC-H (Height) checked, the first dot plot will always default to FSC-A vs FCS-H Note: To make all your plots the same size, select all the plots making sure that the plot of the size you want is selected last and has the yellow sizing boxes on it. Go to the icon in the Worksheet toolbar that says make plots the same size (3 rd icon from the left). 6.4 Optimizing Instrument Settings: Voltage/Threshold/Compensation In the Browser Frame, highlight the Unstained tube (should be tube 1 on your protocol sheet) and position the Acquisition Tube Pointer in the box at the left of the tube Select Low and Run Put the Unstained tube on the SIP and click Acquire in the Acquisition Dashboard Frame In the Cytometer Frame, click on the Parameter Tab and adjust the FSC/SSC voltage to place your population of interest in view on the FSC/SSC dot plot If you are unsure what your population of interest is, you can back gate using one of your positive controls. Revised 1/2016 Page 9 of 26

10 6.4.6 Put on your positive control => draw a gate on the positive population => right click on the FSC/SSC plot => Show Population => select the gate around your positive population => look for the color of your gate in the FSC/SSC plot Adjust the FSC/SSC voltage to place your population of interest on scale So that you don t waste your positive control sample, when your population of interest is in view you can delete the gate and put your unstained tube back on to finish the adjustments Adjust the Threshold in the Cytometer Frame to exclude most of the debris (each tick mark is equal to 10,000 channels) If you are running Kappa beads, the threshold has to be at 5000 or you will not see the beads Draw a Gate around your population of interest (cells or beads) Select all the other plots by holding down the shift key => right click on one plot => Show Population => select the gate in the FCS/SSC plot Using the optimal voltages from the CS&T Report and your single color control tubes, adjust the Voltages of the Fluorescence channels in the Cytometer Frame => Parameter Tab so that you have an optimal voltage while making sure that your positive population is on scale For a weak signal (AB labeling), adjust the voltage to get the best signal to noise ratio For a strong signal (AB labeling), adjust the voltage so that the positive population is on scale After all the voltages have been set, select the Stopping Gate for your target population and the number of Events to Record in the Acquisition Dashboard Frame LEAVE THE STORAGE GATE AT ALL EVENTS!!! You can now setup your experimental panel and name the parameters accordingly using the Experiment Layout tool in the Workspace menu Experiment Menu => Experiment Layout => Label => label your parameters with the correct antibody name and fluorochrome you are using (e.g. CD3-FITC) => ok Put your Unstained tube on the SIP and click Record You can now put the fluidics in High if you choose After the sample is finished recording, remove it from the SIP. Revised 1/2016 Page 10 of 26

11 Expand the unstained tube in the Browser Frame => right click on its Cytometer Settings and Copy Paste the Cytometer Settings into the Global Cytometer Settings under the experiment book by right clicking on the Cytometer Settings => Paste Select any remaining tubes in your specimen => right click and paste the cytometer settings into them as well Click Next Tube to go to the next tube Put your first Compensation tube on the SIP and click Acquire and then Record When the sample if finished recording, remove it from the SIP Put the fluidics in Standby to stop the flow of sheath In your Fluorescent Parameter dot plot, change the parameter on the X axis to the positive control you are working with and the parameter on the Y axis to your next positive control (every time you change a parameter in the plot, you will need to redraw the gates) Click on the Rectangle Gate in the Worksheet Toolbar, and draw it around the Positive population. Make sure it encloses the whole population including those events below axis (use the biexponential view if needed) Draw another Rectangle Gate around the Negative population, making sure that you get those events below the x- and y-axis If you have not already created a Population Hierarchy View and a Statistics View box, do so now Right click on the plot => Show Population Hierarchy Right click on the plot => Create Statistics View The only information you need in the statistics view at this time, are the Populations you are matching up and the Parameters you are looking at with their Means (MFI) To Edit the information you do not need, right click on the Statistics View Box => Edit Statistics => Header Tab => deselect all the header information by unchecking the All box at the top of the column => Populations Tab deselect all the populations you are not looking at as well as the % and # columns by deselecting the box at the top of the column => Statistics Tab select all the fluorescent parameters you are using and their means => OK To compensate your control tubes, you need to match up the Means of the Positive population to that of the Negative Revised 1/2016 Page 11 of 26

12 population of all of the other parameters by adjusting the percent compensation values to add or subtract the Spectral Overlap NOTE: The positive population value should not be smaller than the negative value or be a negative number when compensation is applied because you will be over comping that population In the Browser, highlight the tube you want to compensate (it should turn Blue) => click the Cytometer Settings Tab in the Inspector Frame => click the Compensation Tab and make sure that the Enable Compensation box is checked Make sure that the tube you are trying to compensate is highlighted. If it is not, you will be adjusting compensation for the wrong tube Adjusting the compensation can be done a number of ways. You can use the Arrows to make Small Increments of.10, you can hold down the Control Key and use the Arrows for Medium Increments of 1 or you can Type in the Values for Large Increments Note: I DO NOT suggest using the Slide Ruler, you will be just chasing your tail To help you remember what fluorochrome you need to subtract/add compensation from, look at the top of the compensation table. It has Fluorochrome - % Fluorochrome The positive control will ALWAYS be in the - % Fluorochrome column Rule of Thumb: if unsure what numbers you need to match up in your statistics box, place your thumb over the positive control and match up all the other fluorochrome columns When you finish with the compensation of your first control tube, right click its Cytometer Settings and Copy Spectral Overlap Go up to the Global Cytometer Settings (under your experiment book) and right click and Paste Spectral Overlap With Zeros It is IMPORTANT that you paste with zeros because if you subtract a value to a zero, the software will keep the value Go to your next tube and expand it and Paste Spectral Overlap With Zeros into its cytometer settings Repeat steps thru for the remaining tubes When you are finished with the last compensation tube, Paste the Spectral Overlap With Zeros into the Global Cytometer Settings Revised 1/2016 Page 12 of 26

13 and to all of the previous as well as prior tubes. Click Yes To All. This will guarantee that all tubes have the same cytometer settings. 6.5 Data Recording: Running your Experiment Samples To keep the software from seizing up on you during acquisition, please make up additional experiments for runs with more than 100 samples. If additional experiments are needed to complete the run, after optimizing the first experiment duplicate the experiment without data Put your sample tube on the SIP Click Acquire, wait until the sample gets going and then click Record When the sample is finished recording, remove it from the SIP and click Next Tube to go on to the next sample If you need to Stop Recording before the set number of events have been reached, just click Stop Recording and then Stop Acquiring There are a number of ways to tell when you sample has finished recording: a bell will ding (if not muted), the blue bar in the acquisition control frame will have reached the end and the acquisition tube pointer box as well as the tube in the browser will turn green Repeat steps thru with the rest of your samples Standardizing your Experiments: To standardize your experiments, you need to run a bead after your first experiment to capture where the peaks of your fluorescence parameters fall. This bead should then be the first tube run with future experiments to set your voltages to the mean peak of the first experiment. (Ask Flow Facility staff for help with this approach if needed). 7.0 When finished Running Samples 7.1 Determine if you are, or are not, the last person scheduled to run for that day by looking at the calendar in FacMan. If there is a session behind you, please click on that session to see if the User has put CANCEL in the experiment box IF YOU ARE NOT the last person, then on high run 2 minutes 10% bleach and then 2 minutes dh 2 O => leave the tube of dh 2 O on the SIP => put the fluidics in Low and Standby => top off sheath tank => empty Waste (if necessary) IF YOU ARE the last person, then on high run 5 minutes 10% bleach and then 5 minutes dh 2 O => leave the tube of dh 2 O on the SIP => put the fluidics in Low and Standby => turn off the cytometer => turn off the wet cart (L02 only) => close Diva software and log Revised 1/2016 Page 13 of 26

14 7.2 Exporting Data out of the cytometer controller computer (do not shutdown computer) => top off sheath tank => empty waste (if necessary). When acquisition is completed you need to Export both your Experiment and your FCS files. We also suggest exporting your Experimental Template if you plan on using it more than once If you get an error message when trying to export your data, make sure that the F drive is connected to the controller by clicking My Computer. If F drive says disconnected, click on the Map_Drive icon on the desktop and follow the prompts If F drive still doesn t connect, try rebooting both the Controller computer and the Workstation computer If F drive still fails to connect, export to the local D drive and let the Flow Facility staff know so that they can trouble shoot and transfer your data to the correct drive. 7.3 Exporting Experiment: Right click on the Experiment Book => Export => Export Experiment => OK NOTE: The experiment should export to the default location: F:\BDExport\Experiments If not, Browse => F drive => BDExport => Experiments. 7.4 Exporting FCS Files: Right click on the Experiment Book => Export => FCS => 3.0 format => OK => Save NOTE: The FCS should export to the default location: F:\BDExport\FCS If not, Browse => F drive => BDExport => FCS. 7.5 Exporting Experiment Template: Right click on the Experiment Book => Export => Experiment Template => Type => Select your Initials => Name the Template so that you will know what template it is in the future => Finish. (e.g. 7 color PBMC) NOTE: If you check the Lock Template box, this ensures that the template cannot be overwritten by a template with the same name NOTE: We suggest that you keep a copy of every different template you make up, because to manage the Data Base, we need to delete experiments off of the Browser as well as off of the export drive. These templates stay on the D drive until the User Revised 1/2016 Page 14 of 26

15 leaves the lab or an upgrade to the software is made which will no longer read the older formatted templates. 7.6 Transferring Data to CDMS/Biotrue: After you have exported your Experiment and FCS files, you need to Transfer your FCS folder of data to our Collaborative Data Management System (CDMS)/Biotrue from the Workstation computer Go to the Workstation computer and log in if you haven t already done so (log in information is on the Quick Fact sheet on the wall next to the Workstation computer) Open the Biotrue Uploader located on the desktop and log in (log in information is on the Quick Fact sheet on the wall next to the Workstation computer) Open the BDExport folder located on the desktop => FCS => Days folder => Drag your FCS folder into your Lab s Biotrue dropbox => click Yes, please to upload NOTE: If you are unable to Export your data or transfer it to Biotrue, please contact the Flow Facility via or phone to let us know so that we can assist you NOTE: It is the end Users responsibility to export and upload their FCS files to Biotrue. Facility staff will no longer be checking behind you to make sure you have uploaded your FCS Files NOTE: If you have recorded a LARGE amount of data, please do a property check on your FCS folder to see how big the file is. If it is greater than 500MB, you need to divide the files into two folders before uploading to Biotrue to prevent corruption of any of the files. Please name the second folder the same as the original and put the word cont. at the end. 7.7 Closing FacMan Session: The Fac Man session needs to have the protocol sheet uploaded and the session completed with survey immediately after running the experiment to keep from delaying data management of the Flow Facility staff Update your protocol sheet, and upload it to your Fac Man session BEFORE completing the session End your session in Fac Man and fill out the required survey completely (see attachment #7) NOTE: If you leave ANY question in the survey blank, your session gets red flagged until that question has been answered. Until the survey is completed, the session cannot be moved to invoicing status. So, please remember that you need to go back into that session and answer the question after you have completed the task at hand. Revised 1/2016 Page 15 of 26

16 7.8 If you are the last person, log off of the Workstation computer, please do not shut down the computer, just log off. 7.9 Spray your cooler with 70% Ethanol before leaving the lab Accessing Biotrue in your Lab: URL: Username: flowguest Password: DHVIflow Select DHVI Flow Cytometry Facility and your lab flow data folder 8.0 Attachments 8.1 #1: DHVI Flow Facility Contact List 8.2 #2: Training Checklist / Homework 8.3 #3: Overview of BD LSRII/Fortessa Instruments 8.4 #4: Overview of BD FACSDiva Software 8.5 #5: Cytometer Specific Protocol Form/Template 8.6 #6: Example Job/Session Details Survey 8.7 #7: Troubleshooting Sheath Reservoir Revised 1/2016 Page 16 of 26

17 Attachment #1: DHVI Flow Facility Contact List DHVI Flow Contact List General User Support: FACMAN HELP: MSRBII HELP GHRB HELP Labs/Instruments: A01: GHRB, A02: MSRBII, N01: MSRBII, F01: MSRBII, Bay 5/6 Rm L01: MSRBII, Bay 5/6 Rm L02: MSRBII, Bay 5/6 Rm /29/15 Role Faculty/Staff FacMan Registration Peters Julie Analyst (A01) Cumming Ian Analyst (CHAVI-ID) Jones-Marshall Dawn Analyst (A02) McDermott Patti Scientific Advisor, CHAVI-ID Moody Tony Billing/Finance Hope Tony Facility Director Sempowski Greg Director for Instrumentation Whitesides John Office: Cell: Home: Pager: Revised 1/2016 Page 17 of 26

18 Attachment #2: Training Checklist Prerequisites for Classroom Session! Complete the Introduction to Flow Cytometry web-based training on BD Bioscience s website Will orient the trainee to the basic principals of Flow Cytometry. (~1 hour to complete; required quizzes). 1. URL: 2. Take the quizzes at the end of each section. 3. Record quiz results and bring to Classroom session.! Review the BD FACS Diva Software Overview web-based training on BD Bioscience s website Will orient the trainee to the basic components and function of the acquisition software. (~4 hours to complete). 1. URL: Reference%20Manual.pdf Focus on sections: BD FACSDiva Workspace, Tools for Data Analysis and Data Management.! Print and Review DHVI SOP Flow_010: Policies and Procedures for Flow Facility Users. PDF available on DHVI Flow Facility website: 1. URL: 2. Bring printed copy to Classroom session. Prerequisites for Hands-On Session! Print and Review the appropriate manual: DHVI Flow Facility Training Manual_L01/L02/F01. PDF available on DHVI Flow Facility website: 1. URL: 2. Be sure to review the attachment materials introducing you to the LSRII/LSRFortessa instruments and the Diva Software. 3. Bring printed copy to Hands-On session and Exam session.! Review BD LSRII User's Guide. Please orient yourself with this reference guide and its content. PDF available on DHVI Flow Facility website: 1. URL: Revised 1/2016 Page 18 of 26

19 Attachment #3: Overview of BD LSRII Instrument Figures/text captured from BD LSRII User s Guide See full PDF for details, on Facility website. Components Revised 1/2016 Page 19 of 26

20 Control Panel Revised 1/2016 Page 20 of 26

21 Fluid Control Revised 1/2016 Page 21 of 26

22 Sample Injection Port (SIP) Revised 1/2016 Page 22 of 26

23 Attachment #4: Overview of BD FACSDiva Software Online Course: rence%20manual.pdf Workspace Components Menu Bar Workspace Toolbar Save Browser Frame - Search Field - Browser Toolbar -Folders -Experiments -Specimens -Tubes - Global Instrument Settings - Global Worksheet - Current Tube Pointer Plate Frame Cytometer Frame -Status of the Instrument -Laser Delay/Area Scaling -Instrument Settings for Current Tube Inspector Frame -Experiment Information -Tube Information -Acquisition Information -Instrument Setting Information -Worksheet Information -Plot Information -Plate Information -Keyword Information Worksheet Frame -Worksheet Toolbar -Toggle between Global and Normal Worksheets -Select Arrow -Plots -Magnification Tools -Gating Tools -Editing Tools -Alignment Tools -Population Hierarchy -Statistics View Acquisition Dashboard Frame Revised 1/2016 Page 23 of 26

24 Attachment #5: Cytometer Specific Protocol Form/Template Download CURRENT version from Facility website: Revised 1/2016 Page 24 of 26

25 Attachment #6: EXAMPLE FacMan Job/Session Details Survey Revised 1/2016 Page 25 of 26

26 Attachment #7: Troubleshooting Sheath Reservoir (L02 Only) 1.0 Removing Air from System When The Sheath Reservoir Runs dry: 1.1 Put a tube of dh 2 O on SIP. 1.2 Press prime, and when you see bubble in the water tube press standby (this empties the flow cell). 1.3 While in standby, depressurize the sheath reservoir by loosening the white cap on the reservoir. 1.4 Toggle between prime and run waiting 2 seconds between toggles (repeat a dozen times). 1.5 Put it in standby, and tighten the white cap. 1.6 Wait 40 seconds and then put the machine in run. 1.7 Put the CS&T beads on to make sure that the FSC/SSC are the same as they were during the QC in the morning. If not, repeat the above steps. Revised 1/2016 Page 26 of 26