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43481
FoxP3/Transcription Factor Fixation/Permeabilization Kit
Buffers & Dyes

FoxP3/Transcription Factor Fixation/Permeabilization Kit #43481

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Flow cytometric analysis of mouse splenocytes fixed and permeabilized with #43481 FoxP3/Transcription Factor Fixation/Permeabilization kit, using FoxP3 (3G3) Mouse mAb (PE Conjugate) #49402 (right) and co-stained with CD4-APC #78292, compared to a concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (PE Conjugate) #63630 (left).

To Purchase # 43481S
Product # Size Price
43481S
1 Kit $ 140

Storage:

Store at 4ºC. All components in this kit are stable for at least 6 months when stored at 4ºC.

Protocol

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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 (#46232)
  • 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(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

  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.

Protocol Id: 1744

Product Description

This kit provides the reagents needed to support antibody-based detection of FoxP3 and other transcription factors by flow cytometry. Optimally formulated fixation and permeabilization buffers halt biological activity and enable antibody access to intracellular targets. Antibody dilution, incubation, and wash steps are performed with the included FoxP3/Transcription Factor Permeabilization Buffer, eliminating the need for separate reagents.

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

For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.