Optimizing Instrument Settings for Flow Cytometry
You took great care in the design of your multicolor staining panel, taking into consideration expression levels of your antigens of interest, potential compensation issues…..all those issues you have been trained to consider! Never-the-less, when you actually ran your sample, some of the compensation values were much higher than you expected and the data looked a bit messy – what’s up with that? Using fluorescent beads to calibrate your instrument and set ‘optimal’ PMT voltages does not always translate to ‘optimal’ instrument settings for the particular staining panel you are using. In our example below, the staining panel includes a PerCP-Cy5.5 conjugate (B220) and a PE-Cy7 conjugate (CD3). As can be seen in the figure below, there is some overlap in the emission spectra for these fluorochromes that will have to be dealt with through proper compensation.
Using the settings provided by the bead setup for our instrument, the voltage applied to the PerCP-Cy5.5 detector is 626 and for the PE-Cy7 detector, 669 volts. With this setup, our B220 PerCP-Cy5.5 single stained control looks like this:
PerCP-Cy5.5 Detector and PerCP-Cy5.5 Spillover in PE-Cy7 Detector
YIKES! You know just by looking at this that a significant amount of compensation is going to be required to remove the PerCP-Cy5.5 signal from the PE-Cy7 detector. In fact, that number is 80% and once that is applied, the 2-color plot of B220 PerCP-Cy5.5 vs. CD3 PE-Cy7 looks like this:
Of course, both populations are resolved but there is a lot of spread of the CD3 negative population. By simply decreasing the voltage to the PE-Cy7 PMT to 520, the compensation required to remove the PerCP-Cy5.5 signal from the PE-Cy7 detector is reduced to 12% with excellent resolution of both populations as shown here:
Believe it or not, these data were collected from the same sample of stained cells, the only difference was optimizing the PMT voltage setting for the PE-Cy7 PMT in order to optimize resolution for this particular staining panel!
Some general thoughts:
- Before you collect and save any data, open a screen with a histogram panel for each fluorochrome in your panel. Run each of your single stained controls and evaluate the spillover from that fluorochrome into all of the other open detectors. A good rule of thumb is to be sure that fluorescence in the correct detector is at least half a log brighter than spillover fluorescence in any of the other detectors.
- Using fluorescent beads to calibrate your instrument will provide an excellent starting point for instrument voltage settings but optimizing for your particular panel of reagents will provide excellent data!