Calibration of Becton Dickinson Flow Cytometers for Relative Fluorescence Intensity Measurements
ACCURATE MEASUREMENTS OF FLUORESCENCE INTENSITY SHIFTS CAN BE MADE BY CALIBRATING THE CYTOMETER WITH CHICKEN RED BLOOD CELLS¹
BACKGROUND
Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels.
MATERIALS
1. 10% Bleach solution
2. Double distilled water
3. Unstained cells of the type you are going to analyze in your experiment (negative control sample)
4. Cells stained with monoclonal antibodies of interest (positive control sample)
5. Glutaraldehyde-fixed CRBCs (Biosure, Cat#1004, Riese Enterprise. San Jose, CA)
6. 1x PBS + 2% newborn calf serum + 0.1% Sodium azide (PBSAz)
METHODS
ESTABLISH PMT VOLTAGE SETTINGS FOR YOUR SAMPLES
1. Clean the flow cytometer fluid lines by running 10% bleach for 5 minutes, followed by distilled water for 5 minutes.
2. Launch the CellQuest "Three Color Acquisition Screen" template and set up the software for data acquisition.
3. Put FSC and SSC detectors in LIN mode. Put fluorescence parameter(s) in LOG mode.
4. Set all compensation values to zero.
5. Place the negative-control sample onto the flow cytometer and start acquisition in setup mode.
6. Adjust the FSC and SSC settings so that the cell cluster appears on scale.
7. Position your negative control cluster in appropriate location by adjusting PMT voltages. This is generally within the first decade of the log scale with the cluster fully visible.
8. Put on the positive control sample and make sure the positive signal is not off scale. If so, decrease PMT settings.
9. Make note of final PMT settings for FL-1, FL-2 and FL-3.
DETERMINE CRBC TARGET CHANNELS
10. Put two drops of concentrated CRBC resuspension into a 12x7.5 millimeter tube. Add 1 ml PBSAz and mix well.
11. Open the CellQuest template "CRBC SETUP"² located on the desktop and set up for acquisition.
12. Acquire the CRBCs in setup mode.
13. Adjust the FSC and SSC settings to bring the CRBCs on scale and set all compensation values to zero. Use the PMT voltage settings from #9 above.
14. Position the histogram markers on each peak to calculate the mean channel values for each parameter.
15. Record the mean channel values for each parameter. These are your target channels. Save these instrument settings in an appropriately labeled folder on you disk. Now that your target channels have been determined, you need to calibrate the machine to your own target channels prior to each fluorescence intensity experiment. Note that different lot numbers of CRBCs will yield different target channels; therefore, these need to be re-determined for each lot of CRBCs.
CALIBRATION FOR FLUORESCENCE INTENSITY MEASUREMENTS
16. Clean the machine with bleach and water as described in 1.
17. Prepare the CRBC solution as described in 2.
18. Launch CRBC SETUP template and set up for acquisition. Use instrument settings from #9 (same as #15). Compensation settings must all be zero.
19. Make necessary adjustments to PMT voltages so that the histogram mean channels for each parameter are the same as your target channels (#15). Restart acquisition after each PMT adjustment.
20. Record PMT voltages.
FLUORESCENCE INTENSITY MEASUREMENTS
21. Launch "Three Color Acquisition Screen" and set up for acquisition.
22. Adjust FSC and SSC settings to bring cells on scale.
23. Set PMTs to same voltage as needed to calibrate with CRBCs (#20).
24. Set fluorescence compensation as necessary.
25. Acquire data. Mean channel intensity shifts will be due to receptor expression level changes and not due to instrument variability.
¹This protocol will allow for RFI comparisons only in experiments that utilize the same cells and the same treatments.
²The CRBC Setup document consists of one FSC versus SSC dot plot and one non-gated histogram (with histogram statistics) for each fluorescence parameter. A marker is placed on each histogram to generate accurate mean channel statistics for each parameter. The histograms should have channel values displayed (Plots menu»Log Data Units»Channel Values).
BACKGROUND
Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels.
MATERIALS
1. 10% Bleach solution
2. Double distilled water
3. Unstained cells of the type you are going to analyze in your experiment (negative control sample)
4. Cells stained with monoclonal antibodies of interest (positive control sample)
5. Glutaraldehyde-fixed CRBCs (Biosure, Cat#1004, Riese Enterprise. San Jose, CA)
6. 1x PBS + 2% newborn calf serum + 0.1% Sodium azide (PBSAz)
METHODS
ESTABLISH PMT VOLTAGE SETTINGS FOR YOUR SAMPLES
1. Clean the flow cytometer fluid lines by running 10% bleach for 5 minutes, followed by distilled water for 5 minutes.
2. Launch the CellQuest "Three Color Acquisition Screen" template and set up the software for data acquisition.
3. Put FSC and SSC detectors in LIN mode. Put fluorescence parameter(s) in LOG mode.
4. Set all compensation values to zero.
5. Place the negative-control sample onto the flow cytometer and start acquisition in setup mode.
6. Adjust the FSC and SSC settings so that the cell cluster appears on scale.
7. Position your negative control cluster in appropriate location by adjusting PMT voltages. This is generally within the first decade of the log scale with the cluster fully visible.
8. Put on the positive control sample and make sure the positive signal is not off scale. If so, decrease PMT settings.
9. Make note of final PMT settings for FL-1, FL-2 and FL-3.
DETERMINE CRBC TARGET CHANNELS
10. Put two drops of concentrated CRBC resuspension into a 12x7.5 millimeter tube. Add 1 ml PBSAz and mix well.
11. Open the CellQuest template "CRBC SETUP"² located on the desktop and set up for acquisition.
12. Acquire the CRBCs in setup mode.
13. Adjust the FSC and SSC settings to bring the CRBCs on scale and set all compensation values to zero. Use the PMT voltage settings from #9 above.
14. Position the histogram markers on each peak to calculate the mean channel values for each parameter.
15. Record the mean channel values for each parameter. These are your target channels. Save these instrument settings in an appropriately labeled folder on you disk. Now that your target channels have been determined, you need to calibrate the machine to your own target channels prior to each fluorescence intensity experiment. Note that different lot numbers of CRBCs will yield different target channels; therefore, these need to be re-determined for each lot of CRBCs.
CALIBRATION FOR FLUORESCENCE INTENSITY MEASUREMENTS
16. Clean the machine with bleach and water as described in 1.
17. Prepare the CRBC solution as described in 2.
18. Launch CRBC SETUP template and set up for acquisition. Use instrument settings from #9 (same as #15). Compensation settings must all be zero.
19. Make necessary adjustments to PMT voltages so that the histogram mean channels for each parameter are the same as your target channels (#15). Restart acquisition after each PMT adjustment.
20. Record PMT voltages.
FLUORESCENCE INTENSITY MEASUREMENTS
21. Launch "Three Color Acquisition Screen" and set up for acquisition.
22. Adjust FSC and SSC settings to bring cells on scale.
23. Set PMTs to same voltage as needed to calibrate with CRBCs (#20).
24. Set fluorescence compensation as necessary.
25. Acquire data. Mean channel intensity shifts will be due to receptor expression level changes and not due to instrument variability.
¹This protocol will allow for RFI comparisons only in experiments that utilize the same cells and the same treatments.
²The CRBC Setup document consists of one FSC versus SSC dot plot and one non-gated histogram (with histogram statistics) for each fluorescence parameter. A marker is placed on each histogram to generate accurate mean channel statistics for each parameter. The histograms should have channel values displayed (Plots menu»Log Data Units»Channel Values).
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