Flow Cytometry Technical Resources

Protocols, optimization tips,
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Troubleshooting guides

Troubleshooting guides

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Flow Cytometry Immuno-staining Tips

  • Follow the manufacturers’ instructions as to the storage and handling of the antibodies to avoid degradation and Fc receptor mediated aggregation.
  • Remove antibody aggregates by centrifuging at a high speed at 4°C 5 min. This step is not recommended for IgM antibodies and PE conjugates due to larger sizes.
  • Perform antibody binding reactions on ice and away from direct light.
  • If using adherent cell, do not digest with trypsin as the latter can cleave some surface antigens.
  • In case an antibody has to be diluted and then stored as aliquots, use the staining buffer for dilution. Add 0.09% sodium azide to prevent bacterial contamination.
  • Wash the cells once or twice in staining buffer after each incubation step to remove any unbound antibodies.
  • To amplify the signal for weak antigens, consider using a three step staining process – antigen binding with biotinylated primary antibody → binding with streptavidin conjugated secondary antibody → final binding with anti-streptavidin antibody conjugated to a fluorochrome.
  • Include a viability dye in the antibody cocktail to gate out any dead cells or cell debris.
  • To prevent non-specific Fc receptor staining, add an Fc blocking step or include FBS in the staining buffer. Alternatively, include an isotype control to subtract any signal contributed by the Fc receptor staining.
  • It is always better to acquire the cells soon after staining to minimize any fluorochrome bleaching. In case the cells need to be stored, fix the cells in a suitable fixative and store at 4°C: for overnight storage 0.5% PFA I a good choice but for longer durations like several days or even weeks, the recommended fixative is ethanol.
  • Long term storage in fixative is not recommended as it can significantly increase auto-fluorescence.
  • Antibody titration is recommended to determine the correct concentration of an antibody for the optimum signal. Test different dilutions of the antibody to zero in on the lowest concentration that gives the strongest signal in positive control and the weakest signal in a negative control.

If the specific antibody concentration of a given unpurified antibody preparation is unknown, here are our suggested dilutions for various different sources of antibody:

Tissue culture supernatant

Ascites

Whole antiserum

Purified antibody

1/100

1/1000

1/500

1 µg/mL

 

Keywords: FACS staining protocol, flow cytometry antibody staining procedure, FACS antibody, flow validated

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