Standard Operating Procedure for Setup of BD LSRII Flow Cytometer

Daily Startup

1.Turn on the cytometer; turn on the computer.

2.Fill the sheath tank and verify the following.

• Tank lid is properly sealed.

• Tank is pressurized.

• Tubing is not kinked.

3.Empty the waste tank and verify the following.

• Tank contains sufficient bleach.

• Tubing is not kinked.

4.Purge air bubbles from the sheath filter and sheath line.

• Press Prime twice.

5.Allow the cytometer to warm up.

• 60 minutes for UV applications using 325-nm lasers

• 30 minutes for UV applications using 355-nm lasers

• 30 minutes for all other applications

  1. Set PMTs to the STARTING PMT VALUES (see below for generating these values). Load a sample of mid-range beads (BD Pharmingen #556298) on the sample injection port (SIP). Adjust so that you are acquiring 200-2000 events per second. Gate on the singlet bead population in forward vs. side scatter, and record 10,000 singlet bead events. Record the mean fluorescence, CV, and SD in each fluorescence parameter of the singlet bead population, and save these values in order to trend instrument performance.

Determining Instrument Noise: Perform only once for a given laser + filter configuration

  1. Verify accurate laser delays, reasonable Area Scaling Factor, and minimum sensible Window Extension (typically between 2 and 6. WE impacts noise and calculated PMT values).
  2. Set PMT voltages for each fluorescence parameter initially to 300 volts, and 10000 events into Experiment Layout template.
  3. Load a sample of Rainbow Peak 2 beads (Spherotech #RCP-30-5A-2) on the SIP adjusted so that you are acquiring 200-500 events per second (use the low sample setting).
  4. Adjust the FSC and SSC gain to place the beads reasonably on-scale in FSC-A and SSC-A
  5. Stop Acquisition and use the <Next> button on the Acquisition Dialog to create a Tube for each PMT voltage to be collected (300 to 900 volts every 50 volts = 13 Tubes).
  6. Preset the Instrument Settings for each Tube by right clicking over the Tube name and select <New Instrument Settings> from the popup menu.
  7. Adjust the PMT values for each fluorescence parameter for that Tube and repeat for each Tube to be collected. The correct scatter gains should already be set.
  8. Reset the Acquisition pointer to the first Tube and Acquire data for each Tube using the preset PMT values. It is best to acquire all files over a short time span without stopping or removing the sample.
  9. Analyze the data using a very stringent scatter gate to insure singlet beads ONLY are analyzed (as shown on the right).
  10. Create a Statistics Window to report the CV in each fluorescence Parameter.
  11. Analyze each tube and from the statistics window, record the % CV of the singlet bead population in each fluorescence parameter for each voltage setting. Even a single particle from another run will alter the statistics, and a 2nd region gate drawn around the singlet beads could occasionally be needed to insure only single peak 2 beads are sampled.
  12. Create a graph as shown on the right. Determine the inflection points for each curve, as seen in the example on the right, and record these values as STARTING PMT VALUES for any new experimental setup using this instrument with this configuration of lasers and filters. This value is the PMT voltage you do not want to go below without good reason in order to preserve maximum resolution sensitivity at the low end.

Optimizing on Stained Samples: Perform only once for a given experiment type

  1. Select the appropriate instrument configuration from the “Instrument” menu in FACS Diva software.
  2. Set PMT voltages to the STARTING PMT VALUES as determined in the above section.
  3. Load a sample of cells stained with a complete antibody cocktail from the current experiment, and treated identically as the samples in the experiment (e.g., fixed/permed, etc.). For functional studies, be sure to use a cell sample activated with a positive control, such as SEB, to ensure the existence of positive cells in all possible parameters. Adjust so that you are acquiring about 100-1000 events per second.
  4. Using a forward scatter vs. side scatter dot plot, create a region (P1) corresponding to the cells of interest (e.g., lymphocytes). Then create a histogram plot for each fluorescence parameter, gated on P1.
  5. If there are events in any fluorescence parameter that are reaching the highest fluorescence channel, reduce the corresponding PMT until all events are on-scale.
  6. If the most negative cells in any fluorescence parameter are entirely above the first decade, the corresponding PMT may be decreased to bring the negative cells down to near the expected range of background. Note that this is optional and is intended to provide for more conventional pattern recognition, but if decreased too far this may negatively impact dim population resolution. The high stained background may be lowered until its left hand side begins to visually overlap the right hand side of where unstained was at the STARTING PMT value, but should not go so low as to overlap unstained.
  7. Repeat steps 5 and 6 using samples stained with each antibody cocktail to be used in the current experiment.
  8. Once PMT voltages have been optimized in this way, run a sample of mid-range beads (BD Pharmingen #556298). On a FSC/SSC dot plot, gate on the singlet bead population, and record 10,000 gated events. Write down the mean fluorescence of the singlet population in each fluorescence parameter. Save these values as TARGET VALUES for future runs of this experiment type.

Daily Instrument Calibration: Perform once for each experimental run

1.Load a sample of mid-range beads (Pharmingen #556298) and adjust so that you are acquiring 200-2000 events per second. On a FSC/SSC dot plot, gate on the singlet bead population.

2.While viewing a histogram display of each fluorescence parameter, adjust the PMT voltages until the mean of the singlet population is +/- 10 channels of the target value for that parameter (referring to the TARGET VALUES established for this experiment type, above section, step 8).

3.Prepare compensation controls as follows:

• Choose the appropriate CompBeads for each antibody in the experiment:

Anti-mouse Ig, kappa setPharmingen #552843

Anti-Rat Ig, kappa setPharmingen #552844

Anti-Rat/hamster Ig, kappa setPharmingen #552845

• Dispense a drop of negative control beads and a drop of appropriate CompBeads into a 12x75 mm FACS tube for each fluorescence parameter in the experiment. Label the tubes “FITC control”, “PE control”, etc.

• Add one test of the appropriate antibody to each tube.

• Incubate for 30 minutes at room temperature.

• Wash and otherwise treat the beads in an identical manner as the cells in the experiment, e.g., if cells are fixed and permeabilized, treat the beads with these reagents as well. This is to ensure that procedural effects upon antibody performance are reflected in the compensation controls.

  1. From the “Instrument”—“Instrument Setup” menu in FACS Diva software, choose “Create Compensation Controls”.
  2. Load each compensation control, gate on the singlet bead population in FSC/SSC, and record.
  3. From the “Instrument”—“Instrument Setup” menu in FACS Diva software, choose “Calculate Compensation”. Name the setup file with a name of your choice.
  4. Proceed to acquire your samples using these settings.

Daily Shutdown

1.Install a 12 x 75-mm tube containing 3 mL of a 10% bleach solution on the SIP. Use undiluted BD FACS cleaning solution or bleach diluted in deionized water (DI).

2.Leave the support arm to the side for 1 minute.

3.Place the support arm under the tube, make sure the cytometer is in RUN, and let it run at high flow rate for 5 minutes.

4.Repeat steps 1 through 3 using BD FACS rinsing solution.

5.Repeat steps 1 through 3 using DI water.

6.Leave a tube containing 1 mL of DI water on the SIP with the support arm under the tube.

7.Set the cytometer to standby; turn off cytometer.

8.Shut down the computer.