How does fluorochrome brightness decide which marker it should be paired with?

Why

What matters for detection is how far the positive population sits from the negative one, compared with the spread of the negatives. That separation depends on antigen density, antibody affinity and fluorochrome brightness together. A fluorochrome such as PE or APC gives a high stain index, while FITC or Pacific Blue gives a lower one, though the ranking depends on the instrument's lasers and filters.

The Cossarizza guidelines give the rule directly: match the brightest fluorophores with the lowest-expressed antigens. Their example puts CD33 on APC and CD14 on PE. Brightness is only half of the choice.

A bright dye also spills more light into neighboring detectors, so placing it on an abundant marker that is co-expressed with a dim one can bury the dim marker in spread. Expression level also matters by population. A marker that is bright on monocytes may be dim on the cells you care about.

For example, PD-1 on resting T cells is low, so it earns a bright channel even though it is strong on exhausted cells. Spectral instruments ease detector limits but do not change this logic.