This FOXP3 antibody was purified by affinity chromatography, and conjugated with PE under optimal conditions. The solution is free of unconjugated PE and unconjugated antibody.
Storage & Handling:
The antibody solution should be stored undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
Each lot of this FOXP3 antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For immunofluorescent staining, the suggested use of this reagent is 20 µl per 106 cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.
Application Notes:
Additional reported applications (for the relevant formats) include: Western blotting1, and immunohistochemical staining1 of acetone-fixed frozen sections and formalin-fixed paraffin-embedded sections. The 259D antibody gives strong positivity on paraffin and frozen sections and the antibody stains some epithelial cells. The binding of 206D to FOXP3 can be partially blocked by 259D, but 206D does not show significant blocking effect on 259D binding.
Surface Staining & FOXP3 Buffer Preparation:
Centrifugation steps: perform at 250Xg for 5min Incubation steps: perform at room temperature
1. Perform cell surface staining if necessary (See protocol: Cell Surface Immunofluorescence Staining Protocol). 2. Prepare 1X buffer solutions: The FOXP3 Fix/Perm buffer (4X) must be freshly diluted by diluting one (1) part FOPX3 Fix/Perm buffer (4X) with three (3) parts PBS. The FOXP3 Perm buffer (10X) should be diluted by diluting one (1) part FOXP3 Perm buffer (10X) with nine (9) parts of PBS.
NOTE: The FOXP3 Perm buffer (10X) may have crystalization or precipitation observed when it is stored at 2-8°C, however, it is normal and does not affect the buffer performance. If there is a heavy precipitation observed after diluting to 1X working solution, it may be clarified by filtering. Caution: The FOXP3 Fix/Perm buffer contains paraformaldehyde, which is toxigenic and mutagenic. Please handle with caution and wear gloves, lab coat and necessary protection to avoid direct body contact.
FOXP3 Intracellular Staining Procedures:
3. Add 1 ml of 1X BioLegend's FOXP3 Fix/Perm solution to each tube, vortex and incubate in the dark for 20 minutes, then centrifuge and remove the supernatant. The cell pellet will now be translucent and difficult to see; take care not to dislodge and accidentally aspirate cells at all later stages of staining protocol. 4. Wash: resuspend cells in cell staining buffer (Cat. No. 420201); centrifuge, then discard the supernatant. 5. Wash: resuspend in 1ml 1X BioLegend's FOXP3 Perm buffer; centrifuge, then discard the supernatant. 6. Resuspend cells in 1ml 1X BioLegend's FOXP3 Perm buffer, incubate in the dark for 15 minutes; centrifuge, then discard the supernatant. Resuspend the pellet in 100 µl of 1X BioLegend's FOXP3 Perm buffer. 7. Add appropriate amount of flurochrome conjugated anti-FOXP3 antibody and incubate in the dark for 30 minutes. 8. Wash twice with cell staining buffer (see step 4) then resuspend in 0.5ml cell staining buffer . Analyze with flow cytometer using appropriate instrument settings.
NOTE: BioLegend's FOXP3 Fix/Perm buffer set (Cat. No. 421403) is specifically developed and formulated for intracellular staining FOXP3 with minimum effect on surface fluorochrome staining and is highly recommended for optimal result of FOXP3 intracellular immunofluorescence staining.
Application References:
1. Roncador, G., et al., 2005 Eur. J. Immunol. 35:1681. 2. Yang, Z-Z., et al., 2006. Blood 107:3639. PubMed 3. Gavin, M.A., et al., 2006. PNAS 103:6659. PubMed 4. Groh, V., et al., 2006. Nature Immunology 7:755. PubMed 5. Tran, D. Q., et al., 2007. Blood doi:10.1182/blood-2007-06-094656. PubMed 6.Park, KH., et al. 2008. Cancer Res. 68:8400. PubMed 7. Voo, KS., et al. 2009. Proc Natl Acad Sci U S A. 106:4793. PubMed
Human peripheral blood lymphocytes surface stained with CD4 FITC, then intracellular stained with 259D PE. Quadrant markers were set based on staining with PE mouse IgG1, κ isotype control
Description:
FOXP3 is a 50-55 kD transcription factor, also known as Forkhead box protein P3, Scurfin, JM2, or IPEX. It is proposed to be a master regulatory gene and more specific marker of T regulatory cells than most cell surface markers (such as CD4 and CD25). Transduced expression of FOXP3 in CD4+/CD25- cells has been shown to induce GITR, CD103, and CTLA4 and impart a T regulatory cell phenotype. FOXP3 is mutated in X-linked autoimmunity-allergic dysregulation syndrome (XLAAD or IPEX) in humans and in "scurfy" mice. Overexpression of FOXP3 has been shown to lead to a hypoactive immune state suggesting that this transcriptional factor is a central regulator of T cell activity. In human, unlike in mouse, two isoforms of FOXP3 have been reported: one (FOXP3) corresponding to the canonical full-length sequence; the other (FOXP3 δ2) lacking exon 2. The FOXP3 antibody 259D recognizes human FOXP3 epitope in the region of amino acids 105-235.
Other Names:
Forkhead box protein P3, Scurfin, JM2, IPEX, Zinc finger protein JM2
Structure:
Forkhead/winged-helix transcription factor family, approximately 50 kD, contains zinc finger and forkhead domains
Distribution:
Nuclear; expressed in T regulatory cells
Function:
Transcription factor proposed to be a master regulatory gene in T regulatory cell development and a critical factor for immune homeostasis
Regulation:
FOXP3 is present at high levels in T regulatory cell can also be induced by T cell activation
Interaction:
Interacts with DNA
Antigen References:
1. Hori, S., et al. 2003. Science 299:1057. 2. Fontenot, J.D., et al. 2003. Nature Immunol. 4:330. 3. Ferguson, P.J., et al. 2000. Am. J. Med. Genet. 90:390. 4. Bennett, C.L., et al. 2001. Nature Genet. 27:20. 5. Allan, S.E., et al. 2005. J. Clin. Invest. 115:3276.
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