FoxP3/Transcription Factor Fixation/Permeabilization Kit #43481

FoxP3/Transcription Factor Fixation/Permeabilization Kit #43481

A. Solutions and Reagents

NOTE: Precipitation may occur in some kit components. The presence of precipitate does not affect the performance of the reagent.

Supplied Reagents:

• FoxP3/Transcription Factor Fixation/Permeabilization Diluent (1X) (#58766)
• FoxP3/Transcription Factor Fixation/Permeabilization Concentrate (4X) (#44931): Dilute desired amount to a 1X working solution with FoxP3/Transcription Factor Fixation/Permeabilization Diluent (1X)
• 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.

Additional Reagents (Not Supplied):

• Red blood cell lysis buffer
• Unconjugated or fluorochrome-conjugated primary antibodies
• Fluorochrome-conjugated secondary antibodies (if applicable)
• Cellular dyes (e.g., live-dead discrimination, DNA dyes, etc.), optional. Visit www.cellsignal.com/flowdyes for a full listing of flow cytometry validated cellular dyes.

B. Fixation and Permeabilization

NOTE: Fixable viability dyes such as Ghost Dye™ 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: Add antibodies targeting CD markers or other extracellular epitopes 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.

1. Pellet cells by centrifugation and aspirate 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 ºC). Protect from light.
4. Wash cells 2 times in excess 1X FoxP3/Transcription Factor Permeabilization Buffer.

C. Immunostaining

1. Aliquot desired number of cells into tubes or wells.
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 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 500 µl 1X FoxP3/Transcription Factor Permeabilization Buffer and analyze on flow cytometer.