BD LSRFortessa Flow Cytometer

Starting Up

  1. Turn on the computer. Wait for the computer to boot up.
  2. Log in to the computer (admin, password = BDIS#1) and open BD FACSDiva software. Diva software does not require a username and password, click OK.
  3. Turn on fluidics cart.
  4. Check that the sheath buffer cube has an adequate volume. If it is low replace with an entire new cube. To exchange:
  • Verify that the cytometer is in standby mode.
  • Remove sheath buffer probe from the sheath buffer cube being replaced.
  • Place new sheath buffer cube on to cart.
  • Return sheath buffer probe to new cube and secure it.
  1. Check waste container, empty if necessary and add a small amount of bleach to inactivate pathogens.
  • Verify that the instrument is in standby mode.
  • Disconnect all tubing from cart.
  • Empty the waste container into sink.
  • Add approximately 1L of bleach to the waste container and close it.
  • Reconnect the orange waste tubing and make sure it is not kinked.
  • Reconnect the level sensor line.
  1. Turn on main power switch (green button at right side).
  2. Remove trapped air bubbles from the sheath filter and sheath line, if necessary.
  3. Prime the fluidics 3 times, or as necessary to remove trapped air bubbles from the flow cell.
  4. Wait for 30 minutes for lasers to warm up before running samples.
  5. Watch for instrument connected status at bottom of cytometer window.

 

Checking Cytometer Performance

  1. Select Cytometer > CST. The cytometer disconnects from the BD FACSDiva interface and connects to the CS&T interface.
  2. Verify that the cytometer configuration under the System Summary is the appropriate configuration.
  3. The setup tab indicates the time of the last Performance check for your configuration. If the check was done in the last 24 hours, you do not need to run the beads again. Proceed to step 12 below. If the Cytometer Performance indicates that it has expired, then you will need to run the check. Note that a performance check needs to be run every 24 hours.
  4. Use CST beads. To prepare the beads, mix beads well by inverting the vial or gently vortexing. In a 12x75mm tube, add 0.35mL sheat fluid and 1 drop CST beads. Label the tupe with “CST”, the lot number, the date and the time. Store the bead suspension at 2-25°C in the dark until you are ready to use them.

Notice: Beads are stable at 2°C to 25°C for no more than 20 minutes in direct light, and up to 8 hours if protected from light.

  1. Select the correct bead Lot ID from the menu (bead lot is printed on each Bead vial).
  2. Install tube onto the cytometer loading port.
  3. Under Setup Control, make sure that Check Performance is selected in the Characterize menu.
  4. Click Run. The performance check takes approximately 5 minutes to complete.
  5. Once the performance check is complete, click View Report.
  6. Verify that the performance passed. In the Setup tab, the Cytometer Performance Results should have a green checkbox displayed and the word Passed next to it.
  7. If any parameters did not pass, refer to the concerning technical specialist or the BD Cytometer Setup and Tracking Application Guide for help troubleshooting.
  8. Select File > Exit to close the CST window and connect back to the BD FACSDiva Interface. Click the Use CST Settings, the laser delay, area scaling, and other cytometer settings will be updated to the latest optimized settings from the performance check.

 

Setting Up Your Experiment

  1. Create a new Blank Experiment.
  2. Rename the Experiment (right-click > Rename) using a good descriptive term and the date.
  3. (First time only) In Edit menu > user preferences, select the user preferences that you would like. Generally, this is to clear all preferences in the General tab except Load data after recording.

 

Creating New Application Settings

Application settings are associated with a cytometer configuration and include the parameters needed for the application, area scaling values, PMT voltages, and threshold values, but not compensation. Each time a performance check is run for a configuration, the application settings associated with that configuration are updated to the latest run. Using application settings provides an easy, consistent, and reproducible way to reuse cytometer settings for your commonly used applications.

  1. Start with a new blank experiment (see above)
  2. Select Cytometer Settings in the Browser.
  3. Delete all parameters you will not be using:
    1. In Parameters tab (in Instrument Window) click on small button to left of parameter name
    2. Click delete button (use control key and highlight for multiple deletions)
    3. Repeat for each parameter you are not using.
  4. Click the H and W checkbox to select Height and Width for FCS and SSC to enable doublet discrimination.
  5. Right-click Cytometer Settings in the Browser, then select Application Settings > Create Worksheet. A second global sheet is added with the plots created according to your selections in the Parameter tab. You will use the gray boxes and crosshairs on this worksheet to guide your optimization.
  6. Adjust area scaling factors first, if necessary. This is a more advanced skill, see concerning technical specialist if you feel you need to adjust area scaling factors. For most cells, using the CST Area scaling factors will work fine.
  7. Install the stained cells tube onto the cytometer.
  8. Optimize the FSC and SSC voltages to place the population of interest on scale.
  9. Verify that the positive populations are on scale. If a positive population is off scale, lower the PMT voltage for that parameter until the positive population is entirely on scale. Use gray boxes as a guide when decreasing the PMT voltages. If the negative population is lowered below the gray box, you may decrease your ability to resolve dim populations from the negative population.
  10. Stop acquiring. You do not need to record a data file. Unload the stained cells tube from the cytometer.
  11. You now have your Application settings. To save, right-click Cytometer Settings in the Browser, then select Applications Settings > Save. Name the Application Settings appropriately and click OK. The application settings are saved to the catalog. Application settings do not include compensation settings.

 

Using previously created Application Settings (when you are doing the same experiment again)

  1. In a newly created experiment, ensure that the current CST settings are applied. Then, right-click the Cytometer Settings icon in the Browser and select Application Settings > Apply.
  2. Select your correct previously created Application Settings from the catalog.
  3. Click Overwrite in the dialog that appears.
  4. If a message appears about area scaling, click Yes to accept all changes to cytometer settings.
  5. The parameter list and PMT voltages are updated to match the application settings you previously created.

 

Compensation

  1. Ensure that you have the correct Application Settings applied, or if you are not using them, ensure that you have the correct parameters and PMT voltages for your experiment.
  2. Select Experiment > Compensation Setup > Create Compensation Controls.
  3. Click OK to close the Create Compensation Controls dialog. A compensation controls specimen is added to the experiment, along with an unstained control tube, and a stained control tube for each parameter. Worksheets containing the appropriate plots are added for each compensation tube.
  4. Place the unstained control tube onto the loading port.
  5. Set the current tube pointer to the unstained control tube in the Browser.
  6. Click Load in the Dashboard.
  7. Move the P1 gate to fully incorporate the singlet population.
  8. Right-click the P1 gate and select Apply to all Compensation Tubes.
  9. Click Record Data in the Dashboard to record the events from the unstained control tube.
  10. Unload the unstained control tube.

Notice: do not change the PMT voltages after the first compensation tube has been recorded. To calculate compensation, all tubes must be recorded with the same PMT voltage settings.

  1. Click Next Tube in the Dashboard.
  2. Acquire each compensation tube and record in this manner.
  3. Verify that the snap-to interval gates encompass the positive populations.
  4. Select Experiment > Compensation Setup > Calculate Compensation. If the calculation is successful a dialog appears. Appropriately name the compensation setup.
  5. Click Link & Save to close the dialog box and save the compensation setup and link it to the experiment’s cytometer settings.

 

Recording Sample Data

  1. Create a new specimen in your experiment. If you have a previously created panel template, right click your experiment in the browser and choose New Specimen from the menu. Click the appropriate tab and select the panel template you created previously. Panel templates import worksheets into your experiment also.
  2. If needed, import a blank specimen and create worksheet elements you want to view for this experiment.
  3. Set the current tube pointer at the first tube, install the tube, load it and record it.
  4. Continue for all tubes you wish to run.

 

Shutting Down

  1. Load and run a tube of 10% bleach (FACS Clean) for at least 5 minutes at a HI flow rate.
  2. Repeat with a tube of FACS Rinse.
  3. Repeat with a tube of DI water.
  4. Place the instrument into standby.
  5. Logout of the DIVA software.
  6. If you are the last user of the day, or more than 4 hours until the next user, you will turn the computer, cytometer, and fluidics cart off.

 

Data Export

  1. Select experiment to be exported for CHECKIN in the database.
  2. Select File > Export > Experiments
  3. Browse to choose your own folder.
  4. Click OK.
  5. Transfer your data to a CD/DVD to save it.
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