Controls and gates make a signal interpretable

Spectral overlap means signal from one fluorochrome can enter more than one detector channel. In conventional flow cytometry, matched single-color controls estimate spillover so compensation can transform channel values before biological gates are interpreted. Spectral flow measures the full emission spectrum of each fluorochrome across detectors; unmixing is the mathematical separation of those overlapping spectra using reference controls. It is not simply conventional compensation under another name.

An unstained control shows autofluorescence and background. A matched nonspecific isotype control is an antibody of the same isotype and fluorochrome without the target specificity; its limited purpose is to estimate some nonspecific antibody binding, not to set a universal positive gate or replace an FMO. A single-color control is used for every fluorochrome, including a fluorescent viability dye, when calculating compensation. An FMO control contains all panel reagents except the marker under review and can support a dim, continuous, or spreading boundary.

Dead cells may be more autofluorescent and may bind antibody nonspecifically, so viability is assessed before marker interpretation when viable cells are the intended population. Singlet/doublet discrimination compares pulse area with height or area with width: coincident cells often have disproportionately greater area or altered width at a similar height. Confirm the singlet gate against the validated template and its controls rather than applying a universal gate.