Protocol for FACS Staining

FACS staining and factors to consider and incorporate into your gating

Antibody-stained cell surface markers are often used to identify cell types and delineate the developmental stage of the cell. Additionally, live cells stained with antibodies can be sorted based on their expression of cell surface markers for downstream applications.

To achieve accurate results, it is important to use an optimal staining protocol and gating strategy when assessing cell samples via flow cytometry.



Before You Begin: This is an example protocol. Antibody dilutions and incubation times will need to be optimized on a per antibody basis. Optimization of the antibody dilution and incubation will be essential in minimizing the non-specific binding of the antibody.

General Notes on FACS Staining:

  • Store vials at 2 - 8°C in the dark. Do not freeze fluorochrome-conjugated antibodies.
  • To ensure maximum recovery of antibody volume and proper mixing, quickly centrifuge the vial prior to use and use a pipette to mix the solution; do not vortex antibodies.
  • Antibody-binding kinetics are temperature-dependent. Staining on ice may require longer incubation times. In some instances, certain antibodies may require special incubation conditions that will be noted by the antibody vendor.
  1. Count total cells (including both viable and non-viable cells) obtained after isolation with an EasySep™ kit or other method.
  2. Resuspend viable cells in single-cell suspension using commercially available cell staining buffers or PBS with 2% FBS at a density recommended by the antibody vendor.
  3. Aliquot 100 µL of the cell suspension to each tube or well. Aim to have between 10^5 and 10^6 cells per sample.
  4. Incubate cells with FcR blocking antibodies (anti-CD16/32/64) to reduce non-specific immunofluorescent staining. Alternatively, block cells with serum from the same species as the fluorochrome-conjugated antibody’s host species (the species of the serum may be the same with the host species of either fluorochrome-conjugated primary antibodies or fluorochrome-conjugated secondary antibodies depending on which approach is used).
  5. Add purified primary antibodies or fluorochrome-conjugated primary antibodies at vendor-suggested concentrations. We recommend pre-determining the optimal concentration of antibody to use by titration. The optimal concentration will give you the best resolution while minimizing potential non-specific binding.
  6. Incubate at 2 - 8°C for 30 minutes in the dark, or room temperature for 15 minutes, or 1 hour on ice (refer to antibody vendor’s specifications for details; generally, 30 minutes at 2 - 8°C is the default choice).
  7. Wash cells twice with 2 mL/tube or 200 µL/well of cell staining buffer or PBS with 2% FBS by centrifuging at 350 - 600 x g for 5 minutes at room temperature.
  8. If using purified primary antibodies at step 5, resuspend the washed cell pellet in 100 µL of the buffer with an appropriate anti-species immunoglobulin fluorochrome-conjugated secondary antibody at the vendor-suggested concentrations. We recommend pre-determining the optimal concentration of antibody to use by titration.
  9. Repeat steps 6 and 7.
  10. Stain samples with a viability dye to exclude dead cells at a concentration recommended by the vendor. If fixing the sample for storage prior to data acquisition, we recommend using a fixable viability dye.
  11. Resuspend cells in 100 μL PBS.
  12. Proceed to flow cytometry analysis.
  13. If cells cannot be analyzed immediately, store the cells at 2 - 8°C or on ice in the dark for same-day flow analysis, or fix the cells for next-day flow analysis.

  • Document #PR00029
  • Version 1.0.0
  • November 2020

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