Table 4.

Troubleshooting problems and solutions

Problem	Potential Cause	Potential Solution
No cells on flow cytometer	Cells were knocked out of the plate when performing the wash steps Cytometer is clogged	When decanting supernatant, tap more gently Run 10% bleach or 33% Contrad through the cytometer until the clog is removed
Fluorescent signal in the negative population of the single-color bead control	Non-specific binding of antibody to negative beads	Wash the beads with FACS Staining Buffer again to remove non-specific binding on the negative beads
Spectral signature of single-color control does not match expected signature for the fluorophore	Contamination of control with another fluorescent antibody Tandem dye degraded	Re-prepare control Replace tandem dye with new vial
High proportion of cells are dead	Cells left in DNAse/liberase for too long Cells left in RBC lysing buffer for too long Centrifuged too long and too hard Cells incubated with Protein Transport Inhibitor for too long or too high of a concentration	Reduce the amount of time in the DNAse/liberase Reduce the amount of time in the RBC lysing buffer Reduce the centrifuge speed and/or time Reduce the amount of time or concentration of Protein Transport Inhibitor
Fluorophore spill-over into other channels	Spectra of two markers is indistinguishable	Substitute a different fluorophore-marker pair
Low signal in certain markers	Marker expression might be too low or inexistent Experimental design (i.e., timepoint or vaccine stimulation) does not elicit certain markers Markers chosen were too dim Tandem dyes degraded or decoupled	Check that the marker is supposed to be expressed in the particular cells or animal model Choose a time point closer to vaccination or stimulate the cells with PMA-ionomycin Choose a brighter fluorophore for the specific marker or optimize dilution used Use a new vial of the tandem dye
Not a clear separation in the live/dead sample stain	Residual protein left in the media is binding to and quenching Zombie-NIR Single-color control does not contain both positive and negative populations	Add additional washing step with PBS prior to staining with Zombie-NIR Use ArC Amine Reactive Compensation Bead Kit (Thermo Fisher, cat# A10346) to run single-color live-dead control
Bad separation between negative and positive populations	Antibody concentration is either too high or too low Unbound antibodies were not adequately washed from the samples	Optimize dilution of antibodies Add additional centrifugation and PBS wash