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13399S 100 µl (50 tests) $299.00
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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of murine spleen lymphocytes using FoxP3 (D6O8R) Rabbit mAb (Alexa Fluor® 647 Conjugate) #13399 versus a CD25 antibody. Analysis was performed on a gated CD4+ T cell population.

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Flow Cytometry

A. Solutions and Reagents

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

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. Disaggregate spleens using a 100 µm nylon mesh cell strainer and collect in cold PBS.
  2. Wash cells by centrifugation and resuspend in 5 ml ACK Lysis Buffer for 5 mins. (Red Blood Cell lysis).
  3. Wash by centrifugation in Incubation Buffer.
  4. Resuspend freshly isolated murine spleen cells at 5–10 x 105 cells in 100 µl Incubation Buffer per assay tube.
  5. Add manufacturer's recommended volume of CD25 and CD4 surface marker antibodies to live cells in each sample tube.mix and let incubate for at least 30 min, on ice.
  6. Wash cells by centrifugation and aspirate supernatant.
  7. Resuspend cells in 500 µl of 2% formaldehyde.
  8. Fix for 15 min at room temperature.
  9. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

  1. Add 1ml of 0.3% Triton™ X-100 (v/v in PBS) to the cell pellet.
  2. Vortex and 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 (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 350 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 June 2013

Protocol Id: 62

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 (D6O8R) Rabbit mAb (Alexa Fluor® 647 Conjugate) recognizes endogenous levels of total FoxP3 protein. This antibody recognizes both human and mouse FoxP3 protein.


Species Reactivity: Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro44 of mouse FoxP3 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry analysis in mouse cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated FoxP3 (D6O8R) Rabbit mAb (Flow Preferred) #12653.


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.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
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).
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or outlicensing@lifetech.com.

13399
FoxP3 (D6O8R) Rabbit mAb (Alexa Fluor® 647 Conjugate)