BD LSR Fortessa X20 How To Use - IGC - Facilities...

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Flow Cytometry Service BD LSR Fortessa X20 – How to Use Guide 1 Contents Section 1 Instrument Startup Page 2 Section 2 FACS DiVa setup & automatic compensation Page 3 Section 3 Cleaning procedures, data export & cytometer shutdown Page 6 Section 4 Troubleshooting Page 9 If you have an emergency with your instrument, please contact the Flow Cytometry Staff: From 9AM to 6PM: 214 407 951 After 6PM and weekends: 917 262 274 WARNING Ensure your cells/particles are resuspended in PBS containing NO MORE than 2% serum.

Transcript of BD LSR Fortessa X20 How To Use - IGC - Facilities...

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Flow Cytometry Service BD LSR Fortessa X20 – How to Use Guide

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Contents

Section 1 Instrument Startup Page 2

Section 2 FACS DiVa setup & automatic compensation Page 3

Section 3 Cleaning procedures, data export & cytometer shutdown Page 6

Section 4 Troubleshooting Page 9

If you have an emergency with your instrument, please contact the Flow Cytometry Staff:

From 9AM to 6PM: 214 407 951

After 6PM and weekends: 917 262 274

WARNING Ensure your cells/particles are resuspended in PBS

containing NO MORE than 2% serum.

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Section 1: Instrument Startup

1 The instrument is: OFF (go to step 2); ON (go to step 4).

2 Turn ON the cytometer pressing the green button on its right side. Confirm the computer was OFF before you do this. Otherwise, switch OFF the computer first. Be aware that the lasers take about 15 minutes to warm up and stabilize.

3 Turn ON the computer and screens.

4 Sign in to your Agendo account. Ensure the indication “Agendo connected” was displayed in the dialog window before you sign in.

5 Launch FACS DiVa software and log in your personal area using your username and password.

6 Confirm the PBS tank is full. Otherwise, fill it up with PBS solution taken from the tank labeled “PBS w/ azide” and report the situation through Agendo, using the “Report incident” option.

7 Wait for the indication “Cytometer connected” on the bottom of the Cytometer panel.

8 In the FACS DiVa software select “Use CST Settings”.

IMPORTANT NOTE: The dead volume of BD LSR Fortessa X20 is around 70 µL. For this reason, we recommend to resuspend your samples in a minimal volume of 200 µL. It is strictly forbidden to remove the outer needle of the SIP1. If you believe you need to do it, please contact the facility staff.

1 SIP – Sample Injection Port: the tube/needle that goes inside the FACS tube and through which the sample is aspirated.

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Section 2: FACS DiVa setup & automatic compensation

1 Confirm that all panels are visible: Browser, Cytometer, Inspector, Acquisition Dashboard & Global Worksheet.

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Informative Those windows have the following functions: Browser: to create and manage experiments, specimens and tubes for sample acquisition and recording. Instrument settings may also be copied/pasted, liked/unlinked in this area. Cytometer: to provide information about the cytometer and allow the adjustment of several parameters, such as voltages, compensation, laser delay, thresholds, etc. Inspector: to provide information about and allow the modification of the elements previously selected (e.g. if we select a dotplot, Inspector shows the options to activate the bi-exponential display, to show a grid, to change the background color, etc.; if we select a tube in the Browser panel, Inspector allows to change the tube name, etc. Acquisition Dashboard: controls the sample acquisition and allows to select the number of events shown in the graphs and to be recorded. Global Worksheet: panel where the dotplots and histograms are displayed. It has 2 modes: Global Worksheet (works as a ‘template’ for all tubes and applies every modification to all tubes) and Normal Sheet, which allows individual analysis for each tube, meaning that any change (in a gate, for instance) made in a particular tube will not affect the analysis of the other tubes. To alternate between these two modes one should click in the first button on the top left of this panel.

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3 At the top of the Browser panel, create a New Experiment (click the icon with a book).

4 Create a New Specimen (click the icon with a syringe).

5 Select Tube_001 (inside the syringe) by clicking in the green arrow.

6 Do 3 PRIMEs with the SIP2 arm open and no tube installed. Then set the cytometer to RUN.

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Acquire and record dH2O for 3 minutes in HIGH (name it ‘Initial’). Check in the FSC/SSC dotplot if any events appear. If you get too many events per second and/or falling into your gates, replace the dH2O tube by a new one prepared by you. If the problem persists, perform the cleaning procedure as indicated in Section 3 and report the situation through Agendo, using the “Report incident” option.

8 If the machine is clean, you can start acquiring your samples (Step 21) or perform Automatic Compensation (Step 9).

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To perform automatic compensation, do to Experiment Menu à Compensation Setup à Create Compensation Controls. This will open a window entitled Create Compensation Tubes. Confirm the detectors displayed are the ones you will need. Otherwise, delete the ones you won’t use. If you do not have an Unstained Control, unselect the option “Add Separated Unstained Control”. Click OK.

10 In the Acquisition Dashboard panel choose to display 500 events.

11 In the Browser panel, scroll down the tubes hidden under the Compensation Control Specimen and select the unstained tube by clicking in the green arrow.

12 In the Worksheet window, select all histograms with the aid of the Shift key. Then go to the Inspector panel and select “grid” to visualize four quadrants in all histograms.

2 SIP – Sample Injection Port: the tube/needle that goes inside the FACS tube and through which the sample is aspirated.

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13 Install the unstained control sample in the cytometer and set the speed to LOW. Start acquisition in the Acquisition Dashboard.

14 In the Cytometer panel, in the Parameters tab, adjust the voltages of FSC and SSC in order to roughly place your cells of interest at the center of the dotplot.

15 Adjust P1 gate around your population of interest. Then, right click in the mouse and select “Apply to All Compensation Controls”.

16 In the Cytometer panel, in the Parameters tab, adjust the voltages of each fluorescence detector so the negative cells sit in the left quadrant of the histograms, touching the right border. Before recording each control, confirm that each fluorochrome is the brightest in its respective channel.

17 Record the unstained tube and click Next Tube.

18 Install the first Single Staining Control tube in the cytometer. Record all the events and adjust the P2 region around the positive population. Click Next Tube.

19 Repeat the previous step for all Single Staining Controls.

20 Go to Experiment Menu à Compensation Setup à Calculate Compensation and select Link & Save.

21 In the Browser panel select Tube_001 by clicking the green arrow.

22 In the Cytometer panel open the Parameters tab and select the detectors/fluorochromes that you will not use with the aid of the shift key. Delete them by pressing the button at the bottom.

23 In the Worksheet window choose between the option of Global Worksheet or Normal Worksheet according to your purpose. We recommend using Global Worksheet for acquisition.

24 In the Cytometer panel select “W” (in addition to “A”) in the FSC parameter. This will be useful in case you need to exclude doublets.

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In the Worksheet window create a dotplot FSC-A versus SSC-A to select your primary population – P1; and a dotplot FSC-A versus FSC-W to exclude doublets – P2. To create a gate inside another, right click in the mouse while touching a dotplot and select Population Hierarchy, which will open a new window with that information. In that window select the region you would like to have as parent gate and create a new gate. Add all the dotplots and histograms you will need for your analysis and draw the draft of the gates in each one of them.

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26 In the Experiment menu select “Experiment Layout” and write the name of the molecules you are analyzing in the corresponding fluorochrome/channel.

27 In the Acquisition Dashboard panel select the number of events to visualize and to record, as well as the gate inside which the events should be counted and recorded.

28 You are set to start your acquisition and record your samples. Be aware of the volume aspirated during acquisition to avoid air to enter the cytometer.

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Section 3: Cleaning procedures, data export & cytometer shutdown

1 If you have used propidium iodide (PI) or other viability dyes go to step 2. If you did not use PI or other viability dye staining in your experiment, go to step 8.

2 If you have an extra sample with cells not labelled with PI at relatively high concentration, go to step 3. If you don’t, go to step 4.

3 Perform a 3-minute acquisition of your PI-unlabeled concentrated cells in HIGH. Record this acquisition (name it ‘Unstained to Clean PI’). At the end go to step 8.

4 Install a tube of bleach with the SIP arm open for 30 seconds.

5 Close the SIP arm and aspirate bleach in Super HIGH for 5 minutes. Record this acquisition (name it ‘Bleach’).

6 Install a tube of detergent with the SIP arm open for 30 seconds.

7 Close the SIP arm and aspirate detergent in Super HIGH for 10 minutes. Record this acquisition (name it ‘Detergent’). At the end, go to step 12.

8 Install a tube of bleach with the SIP arm open for 30 seconds.

9 Close the SIP arm and aspirate bleach in Super HIGH for 5 minutes. Record this acquisition (name it ‘Bleach’).

10 Install a tube of detergent with the SIP arm open for 30 seconds.

11 Close the SIP arm and aspirate detergent in Super HIGH for 5 minutes. Record this acquisition (name it ‘Detergent’).

12 Install a tube of dH2O with the SIP arm open for 30 seconds.

13 Close the SIP arm and aspirate dH2O in Super HIGH for 5 minutes. Record this acquisition (name it ‘H2O’).

14 Set the cytometer in Standby.

15 Refill the sheath tank with PBS from the “PBS w/ azide” container.

16 Export your Experiment from FACS DiVa by right clicking on the Experiment name with the mouse and selecting Export à Experiment. Select the local destination folder for temporarily saving your files (usually there is a folder in the Desktop).

17 DELETE your experiment from DiVa. If you wish to keep your settings and page analysis, copy the Experiment and paste it in DiVa without data.

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18 Copy the exported files to your personal directory in the remote server by launching Fileszilla from the Start Menu. You'll be prompted for your username and password and after that just need to select the folder you want to access and drag your local files into the remote folder.

19 After transferring your data to the remote server, delete the files from the local folder.

20 Quit DiVa (Menu File → Quit).

21 Check if there is a booking after you in the next 3 hours. If YES, go to step 22. If NO, go to step 23.

22 Leave the cytometer in Standby with dH2O on the SIP and log off from Agendo. Remove your gloves, tubes and other belongings and clean up any trash. You are done.

23 Switch OFF the cytometer in the green button on its right side.

24 Log off from Agendo and turn the computer off. Remove your gloves, tubes and other belongings and clean up any trash. You are done.