Cellular autofluorescence is caused by FAD and FMN (515 nm emission), NADH (460 nm emission), etc. Such autofluorescence hinders quantitative detection of low levels of antibody binding.

For fixed cells, a 30 min wash in PBS/0.1% sodium borohydride significantly reduces such fluorescence.

A 10 min incubation at 4° with ice-cold, 1ug/ml trypan blue solution will reduce autofluorescence after 488 nm excitation (used with FITC-labeled probes).

Noise is basically the signal seen in the negative-control sample (no primary antibody, etc.). Any fluorophore conjugate is good for an antigen expressed at a high level. However, for lower expression levels, a better signal-to-noise ratio may be achieved by switching fluorophores.

On FACS Calibur machine, PE is better than APC > PerCP > FITC.

Note that PerCP cannot be used with high laser power in machines like the Vantage (>150 mW).

Antibody aggregates increase non-specific staining. Reduce by proper handling and storage. A high speed spin x 10 min at 4° (not for IgM or PE-conjugated antibodies - heavy) can be used to remove aggregates.

Non-specific binding may be reduced by pre-incubating (before the secondary antibody incubation) cells with serum or purified, but unlabeled, IgG of same species (e.g., goat serum if using a goat secondary antibody). Usually, incubation for 10 min with 10ug IgG per 10e6 cells is enough.

Anti-Fc receptor antibodies can also be used to block non-specific binding of fluorophore-conjugated primary antibodies - 0.5 ug per 10e6 cells, as above.

Dead cells non-specifically bind antibodies; so, minimize cell death - use cells right away after harvest, keep at 4°, include BSA or heat-inactivated serum in all buffers, analyse stained cells right away, etc.

Dead cells can be excluded from analysis by gating using stains such as propidium iodide (FL3 channel; overflow into FL2, but compensation is not needed even though you are using FL2 for antigen detection using, say, the PE fluorophore.)