1. Be sure that all the samples used for flow cytometry are single cell suspension in case of block of apparatus by cell aggregates and fragments.
2. The concentration of single cell suspension should reach 1×106/ml, or too low concentration may have bad effect on the results.
3. Suitable methods should be made for different samples to be disposed in order to obtain single cell suspension, such as mechanically disjointed, enzymatic discomposition, and so on.
4. Avoid death cells producing by personal equation. If the content of death cells > 20%, then the results would be unreliable.
1. The antibody coupled with fluorescence dyes should be preserved at 4℃ in dark.
2. Blocking reagents such as 0.5% BSA and 1% fetal bovine serum should be used to block non-specific binding sites.
3. The sample stained by fluorescence dyes should be kept away from lights to make sure its stability.
4. The buffer used for washing cells after staining should also be added with 0.5% BSA and 1% fetal bovine serum, not only for blocking non-specific binding sites, but also for maintain the activity of cells.
5. Adequate washing should be done to avoid false-positive and false-negative.
6. Intese mixing should be avoided in case of producing too many broken cells which may result of non-specific flurescence.
7. Suitable speed of centrifugation should be set to decrease the content of adhered cells.
Reduce cellular autofluorescence
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.
10 min incubation at 4℃ with ice-cold, 1 μg/ml trypan blue solution will reduce autofluorescence after 488 nm excitation (used with FITC-labeled probes).
Right conjugate to improve signal-to-noise ratio
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).
Reducing non-specific secondary antibody staining
Antibody aggregates increase non-specific staining. Reduce by proper handling and storage. A high speed spin × 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 10 μg IgG per 106 cells is enough.
Anti-Fc receptor antibodies can also be used to block non-specific binding of fluorophore-conjugated primary antibodies - 0.5 μg per 106 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, analyze 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.)