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To Purchase # 12653S

12653P 40 µl (20 tests) $139.00
12653S 100 µl (50 tests) $269.00


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  • Additional protein information
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H M Endogenous 45 Rabbit IgG

Flow Cytometry

Flow cytometric analysis of murine spleen lymphocytes using a CD25 antibody and FoxP3 (D6O8R) XP® Rabbit mAb. Anti-rabbit IgG (H+L), (F(ab')2 ) Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary 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 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

FoxP3 (D6O8R) XP® Rabbit mAb 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.

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.

FoxP3 (D6O8R) XP® Rabbit mAb