Flow Cytometry, FoxP3/Transcription Factor Fixation and Permeabilization Protocol

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our FoxP3/Transcription Factor Fixation/Permeabilization Kit #43481, or individually using the catalog numbers listed below.

1. FoxP3/Transcription Factor Fixation/Permeabilization Diluent (1X) (#58766)
2. FoxP3/Transcription Factor Fixation/Permeabilization Concentrate (4X) (#44931): Dilute desired amount to a 1X working solution with FoxP3/Transcription Factor Fixation/Permeabilization Diluent (1X)
3. FoxP3/Transcription Factor Permeabilization Buffer (10X) (#68751): Dilute desired amount to a 1X working solution with reverse osmosis deionized (RODI) or equivalent grade water. Use for all wash steps and antibody incubation following fixation.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. View our listing of cellular dyes validated for use in flow cytometry.

B. Fixation and Permeabilization

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation. If using whole blood, lyse red blood cells using RBC Lysis Buffer (#46232) and wash by centrifugation prior to fixation.

NOTE: Fixable viability dyes such as Ghost Dye(TM) Violet 510 Viability Dye #59863 should be added prior to fixation, following the dye product recommended protocol. Proceed with fixation once excess dye has been removed.

NOTE: Antibodies targeting CD markers or other extracellular epitopes may be added prior to fixation. The antibodies will remain bound to the target of interest during the fixation process. A wash step prior to fixation may be performed but is not necessary.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

1. Pellet cells by centrifugation and remove supernatant.
2. Resuspend cells in 1 mL FoxP3/Transcription Factor Fixation/Permeabilization 1X working solution, prepared as described above. Mix well to dissociate pellet and prevent cross-linking of individual cells.
3. Incubate 30-60 min at room temperature (20-25°C). Protect from light.
4. Wash by centrifugation in excess 1X FoxP3/Transcription Factor Permeabilization Buffer. Discard supernatant. Repeat.

C. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

1. Aliquot desired number of cells into tubes or wells. (Generally, 5x10⁵ to 1x10⁶ cells per assay).
2. Resuspend cells in 100 µl of diluted antibody, prepared in 1X FoxP3/Transcription Factor Permeabilization Buffer at the recommended dilutions. See individual antibody datasheets or product webpage for recommended dilution, or determine via titration.
3. Incubate for 1 hr at room temperature (20-25°C). Protect from light.
4. Wash by centrifugation in excess 1X FoxP3/Transcription Factor Permeabilization Buffer. Discard supernatant. Repeat.
5. If using a fluorochrome-conjugated primary antibody, resuspend cells in 200-500 µl 1X FoxP3/Transcription Factor Permeabilization Buffer and analyze on flow cytometer; for unconjugated primary antibodies, proceed to next step.
6. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in 1X FoxP3/Transcription Factor Permeabilization Buffer at the recommended dilution.
7. Incubate for 30 min at room temperature (20-25°C). Protect from light.
8. Wash by centrifugation in excess 1X FoxP3/Transcription Factor Permeabilization Buffer. Discard supernatant. Repeat.
9. Resuspend cells in 200-500 µl 1X FoxP3/Transcription Factor Permeabilization Buffer and analyze on flow cytometer.