Methods for optimizing PMT voltages on BD digital instruments
Shortcut option 1
The first option is to use the CS&T (Cytometer Setup &Tracking) voltages that were previously determined by FMCF staff using the BD CS&T software. The principles used to derive the CS&T targets are similar to those in the Minimal Noise protocol described here because both approaches determine the voltage needed to place dim particles above the level of electronic noise. However, the intrinsic CVs and optical properties of the cells are unlikely to be the same as the beads, so the CS&T PMT voltages may not be truly optimal for your application. Nonetheless, they provide reasonable starting PMT voltages for many applications.
By default when a new Experiment is created, it is automatically set to the CS&T voltages (the purple dot beside the cytometer setting icon in the software browser window indicates cytometer settings are set to CS&T voltage settings - once you alter any of the voltages the purple dot will disappear). To apply the current Cytometer Setup &Tracking voltages to an existing experiment, do the following:
- In the experiment, right-click the experiment-level Cytometer Settings icon and choose "Apply Current CST Settings". (If you see a warning message that the Cytometer Setup and Tracking settings have expired, click "OK" anyway.)
Shortcut option 2
The second option is to use the "Lymphocyte Minimum Target Values" that the FMCF has established. These target values apply only to the filters in the default ("New Advanced") configuration. Fresh ex vivo lymphocytes are commonly used to optimize PMT voltages for flow cytometers because they have measurable (but dim) autofluorescence in many (though not all - see above) wavelengths. The Lymphocyte Minimum Target Values were determined using fresh ex vivo murine lymphocytes as described below. Adjusting each PMT voltage to achieve these bead targets will ensure that lymphocytes (and other cell types that have similar or greater amounts of autofluorescence) will be at a gain where electronic noise makes a minimal contribution to the measured signal.
In our experience, the CS&T voltages versus the Lymphocyte Minimal Targets yield very similar MFIs for unstained lymphocytes in the blue-green-yellow-orange detectors. However, we (and BD) have found that the CS&T method consistently yields much higher MFIs (often >100) for unstained lymphocytes and dim beads in the red and far-red detectors. Although both methods produce PMT voltages that provide reasonable resolution sensitivity, we prefer to use the Lymphocyte Minimum Bead Target values so that unstained cells are not placed too high on scale in the red and far-red channels.