It is generally recommended that PMT voltages are optimized to maximize the performance of any new flow cytometry assay. However, the voltage settings obtained from the initial voltration should only act as a baseline when the assay is later repeated in longitudinal studies or assay is performed on a different instrument. To standardize data between experimental runs (or instruments) bright fluorescent multicolor beads or aliquoted biological reference sample (e.g. frozen PBMC pool from healthy donors) are used to calibrate the measured signal intensities in each detector. In short, follow the steps below:
- Perform initial voltage optimization (preferably using actual experimental samples) to maximize assay performance.
- Run bright beads (or reference sample) with these baseline voltages and record their fluorescence intensities on each channel. These are your target values in all subsequent assay repeats.
- In each future experiment, start by running the calibration beads and adjusting PMT voltages as needed to obtain the target values. This minimizes data fluctuations due to day-to-day differences in instrument performance.
- Run your assay and remember to include all the relevant controls.