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PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools


H Endogenous Rabbit IgG

Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells using FoxP3 (D6O8C) Rabbit mAb (Alexa Fluor® 488 Conjugate) versus an anti-human CD25 antibody, gated on either CD4+ T cells (right) or CD4- T cells (left).

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Flow Cytometry

A. Solutions and Reagents

NOTE: Prepare solutions with RODI (reverse osmosis deionized) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. Formaldehyde (methanol free).
  3. 2% Formaldehyde (stock formaldehyde diluted in PBS; make fresh on day of experiment).
  4. Triton™ X-100.
  5. Ficoll-Paque™.
  6. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9988) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: Surface staining of CD4 and CD25 antibodies should be performed on live cells prior to fixation/permeabilization, as per manufacturer’s requirements.

  1. Isolate PBMCs from whole blood by Ficoll density centrifugation as per manufacturer’s instructions.
  2. Wash 2x by centrifugation using Incubation Buffer.
  3. Resuspend cells in Incubation Buffer and aliquot at a concentration of 1 x 106 cells/100 µl/sample tube.
  4. Add CD4 and CD25 antibodies to assay tubes as per manufacturer’s recommended volume or concentration and incubate for 30 min. on ice.
  5. Add 2 ml of Incubation Buffer and wash by centrifugation.
  6. Aspirate supernatant and resuspend cells in 500 µl of 2% formaldehyde.
  7. Fix for 15 min at room temperature.
  8. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

  1. Resuspend cells in 1 ml of 0.1% Triton™ X-100 (v/v in PBS).
  2. Let stand for 30 min at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.

D. Immunostaining

  1. Resuspend cell pellets in 100 µl of FoxP3 (D6O8C) XP® Rabbit mAb #12632 or FoxP3 (D6O8R) XP® Rabbit mAb #12653 working solution (stock antibody diluted 1:200 in Incubation Buffer).
  2. Incubate for 1 hr at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.
  4. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in Incubation Buffer at the recommended dilution.
  5. Incubate for 30 min at room temperature.
  6. Wash 2X by centrifugation in Incubation Buffer.
  7. Resuspend cells in 500 µl of Incubation Buffer and analyze on flow cytometer.
  8. FoxP3 can be plotted against CD25 on a bivariate scattergram gated on CD4+ T lymphocytes.

posted July 2013

revised October 2013

Protocol Id: 63

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

FoxP3 (D6O8C) Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total FoxP3 protein.

Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro293 of human FoxP3 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated FoxP3 (D6O8C) Rabbit mAb #12632.

Forkhead box (Fox) proteins are a family of evolutionarily conserved transcription factors containing a sequence known as Forkhead box or winged helix DNA binding domain (1). The human genome contains 43 Fox proteins that are divided into subfamilies. The FoxP subfamily has four members, FoxP1 - FoxP4, which are broadly expressed and play important roles in organ development, immune response and cancer pathogenesis (2-4). The FoxP subfamily has several characteristics that are atypical among Fox proteins: their Forkhead domain is located at the carboxy-terminal region and they contain motifs that promote homo- and heterodimerization. FoxP proteins usually function as transcriptional repressors (4,5).

FoxP3 is crucial for the development of T cells with regulatory properties (Treg) (6). Mutations in FoxP3 are associated with immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX) (7), while overexpression in mice causes severe immunodeficiency (8). Research studies have shown that FoxP3 functions as a tumor suppressor in several types of cancer (9-11).

1.  Myatt, S.S. and Lam, E.W. (2007) Nat Rev Cancer 7, 847-59.

2.  Shu, W. et al. (2001) J Biol Chem 276, 27488-97.

3.  Lu, M.M. et al. (2002) Gene Expr Patterns 2, 223-8.

4.  Koon, H.B. et al. (2007) Expert Opin Ther Targets 11, 955-65.

5.  Li, S. et al. (2004) Mol Cell Biol 24, 809-22.

6.  Ochs, H.D. et al. (2007) Immunol Res 38, 112-21.

7.  Bennett, C.L. et al. (2001) Nat Genet 27, 20-1.

8.  Kasprowicz, D.J. et al. (2003) J Immunol 171, 1216-23.

9.  Zuo, T. et al. (2007) Cell 129, 1275-86.

10.  Zuo, T. et al. (2007) J Clin Invest 117, 3765-73.

11.  Wang, L. et al. (2009) Cancer Cell 16, 336-46.

Entrez-Gene Id 50943
Swiss-Prot Acc. Q9BZS1

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.
The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).

FoxP3 (D6O8C) Rabbit mAb (Alexa Fluor® 488 Conjugate)