Biolegend, 320403, True-Nuclear™ Human Treg Flow Kit, 25tests

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 259D antibody recognizes human FOXP3 epitope in the region of amino acids 105-235.
25tests
Verified Reactivity: Human
Reported Reactivity: Cynomolgus, Rhesus, Baboon, Chimpanzee
Antibody Type: Monoclonal
Immunogen: Full-length FOXP3 protein
Concentration: Lot-specific (to obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.)
Storage & Handling: This kit is guaranteed for six months. Upon receipt, store between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application: ICFC - Quality tested
Application Notes: Materials Provided: 1. Alexa Fluor® 488 anti-human FOXP3 - 25 tests 2. Alexa Fluor® 488 Mouse IgG1, κ isotype control - 25 tests 3. True-Nuclear™ Transcription Factor Buffer Set - 120 tests (Cat. No. 424401) 4. anti-human CD4 PE-Cyn5/CD25 PE Cocktail - 50 tests Materials Not Included: 1. Cell Staining Buffer (Cat. No. 420201) 2. Single color compensation controls Immunofluorescence Staining Procedures: 1. Perform cell surface staining as described in BioLegend's Cell Surface Immunofluorescence Staining Protocol. Add 20 µL of the anti-human CD4 PE-Cyn5/CD25 PE cocktail to each tube and incubate in the dark for 20 minutes. 2. Add 2 mL of the cell staining buffer, centrifuge tubes at 400 x g at room temperature for five minutes, and discard the supernatant. 3. Repeat Step 2, for a total of two washes. 4. Add 1 mL of the Transcription Factor 1X Fix solution to each tube, vortex, and incubate at room temperature in the dark for 45-60 minutes. 5. Without washing, add 2 mL of the Transcription Factor 1X Perm Buffer to each tube. 6. Centrifuge tubes at 400 x g at room temperature for five minutes, and discard the supernatant. 7. Add 2 mL of the Transcription Factor 1X Perm Buffer to each tube. 8. Centrifuge tubes at 400 x g at room temperature for five minutes, and discard the supernatant. 9. Resuspend the cell pellet in 100 µL of the Transcription Factor 1XPerm Buffer. 10. Add 5 µL of Alexa Fluor® 488 anti-human FOXP3 antibody or 5 µL of Alexa Fluor® 488 mouse IgG1, κ isotype control into the appropriate tubes. Incubate in the dark at room temperature for at least 30 minutes. 11. Add 2 mL of the Transcription Factor 1X Perm Buffer to each tube. 12. Centrifuge tubes at 400 x g at room temperature for five minutes, and discard the supernatant. 13. Add 2 mL of the cell staining buffer. 14. Centrifuge tubes at 400 x g at room temperature for five minutes, and discard the supernatant. 15. Resuspend in 0.5 mL cell staining buffer and then acquire tubes on a flow cytometer. Caution: The True-Nuclear™ Transcription Factor Buffer Set contains paraformaldehyde, which is toxic and mutagenic. Please handle with caution. Wear gloves, lab coats, and necessary protection to avoid direct contact. NOTE: For flow cytometric staining with this clone, True-Nuclear™ Transcription Factor Buffer Set (Cat. No. 424401) offers improved staining and is highly recommended.
Application References(PubMed link indicates BioLegend citation): Roncador G, et al. 2005 Eur. J. Immunol. 35:1681. Yang ZZ, et al. 2006. Blood 107:3639. PubMed Gavin MA, et al. 2006. P. Natl. Acad. Sci. USA 103:6659. PubMed Groh V, et al. 2006. Nature Immunology 7:755. PubMed Tran DQ, et al. 2007. Blood doi:10.1182/blood-2007-06-094656.PubMEd Long SA, et al. 2008. J Autoimmun. 30:293. PubMed Gong G, et al. 2009. Blood 113:837. PubMed Long SA, et al. 2009. Eur J. Immunol. 39:612. PubMed Long SA, et al. 2010. Diabetes. 59:407. PubMed Ferraro A, et al. 2014. PNAS. 111:1111. PubMed Vudattu NK, et al. 2014. J Immunol. 193:587. PubMed Dupont G, et al. 2014. Cytokine. 69:146. PubMed
RRID: AB_3097009 (BioLegend Cat. No. 320403)
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
Interaction: Interacts with DNA
Cell Type: Tregs
Biology Area: Cell Biology, Immunology, Transcription Factors
Molecular Family: CD Molecules, Nuclear Markers
Antigen References: Hori S, et al. 2003. Science 299:1057.
Regulation: FOXP3 is present at high levels in T regulatory cell can also be induced by T cell activation
Gene ID: 50943
UniProt: View information about FOXP3 on UniProt.org
Clone: 259D
Regulatory Status: RUO
Other Names: Forkhead box protein P3, Scurfin, JM2, IPEX, Zinc finger protein JM2
Q: Can I stain whole blood with anti-FOXP3 using your Foxp3 staining kit?
A: It is not recommended. It is best to use PBMCs for this testing.