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Safety Data Sheet (SDS) Alexa Fluor 488 Anti-mouse/rat/human FOXP3     Product Data Sheet (PDF)    
Alexa Fluor® 488 Anti-mouse/rat/human FOXP3
2200055 25 tests $155.00       
2200060 100 tests $310.00       
Clone: 150D
Isotype: Mouse IgG1, κ
Reactivity: Human, Mouse, Rat, Cross-Reactivity: Cynomolgus, Rhesus, Baboon
Immunogen: Full-length FOXP3 protein
Formulation: Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and 0.2% (w/v) BSA (origin USA).
Preparation: The antibody was purified by affinity chromatography, and conjugated with Alexa Fluor® 488 under optimal conditions.
Concentration: Lot-specific (please contact technical support for concentration and total µg amount, or use our Lookup tool if you have a lot number.)
Storage & Handling: The FOXP3 antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
Application:

ICFC - Quality tested

Application Notes:

Additional reported applications (for the relevant formats) include: Western blotting1.

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.
2. Prepare 1X buffer solutions of FOXP3 Fix/Perm buffer and FOXP3 Perm buffer in 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 FOXP3 Fix/Perm solution to each tube, resuspend the cells (gentle vortex) and incubate at room temperature 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; centrifuge, then discard the supernatant.
5. Wash: resuspend in 1ml 1X FOXP3 Perm buffer; centrifuge, then discard the supernatant.
6. Resuspend cells in 1ml 1X FOXP3 Perm buffer, incubate in the dark for 15 minutes; centrifuge, then discard the supernatant. Resuspend the pellet in 100 µl of 1X FOXP3 Perm buffer.
7. Add appropriate amount of flurochrome conjugated anti-FOXP3 antibody and incubate at room temperature in the dark for 30 minutes.
8. Wash twice with cell staining buffer (see step 4) then resuspend in 0.5 ml cell staining buffer. Analyze with flow cytometer using appropriate instrument settings.

NOTE: FOXP3 Fix/Perm buffer set 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.

Recommended Usage:

Each lot of this antibody is quality control tested by intracellular flow cytometry using our True-Nuclear™ Transcription Factor Staining Protocol. For flow cytometric staining, the suggested use of this reagent is 5 µl per 106 cells in 100 µl volume. It is recommended that the reagent be titrated for optimal performance for each application.

* Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm.

Application References:

1. Roncador G, et al. 2005 Eur. J. Immunol. 35:1681.
2. Mayack. S,et al. 2006. J. Immunol.176:2059. J. Immunol
3. Yang ZZ, et al. 2006. Blood 107:3639.
4. Gavin MA, et al. 2006. P. Natl. Acad. Sci. USA 103:6659.
5. Groh V, et al. 2006. Nature Immunology 7:755.
6. Lewkowicz P, et al. 2006 J. Immunol. 177:7155.
7. Luke PPW, et al. 2006. Amer. J. Transplant. 6(9):2023.
8. Bamias G, et al. 2007. J. Immunol. 178:1809.
9. Valencia X, et al. 2007. J. Immunol. 178:2579.PubMed
10. Davidson TS, et al. 2007. J. Immunol. 178:4022.
11. MacDonald K PA, et al. 2007. Blood doi:10.1182/blood-2007-01-067249.
12. Jaffar Z, et al. 2007. J. Immunol. 179:6193.
13. Müller M, et al. 2007. J. Immunol. 179:2774.
14. Jordan JM,et al. 2008.Infect Human. 76:3717. PubMed
15.Golovina TN,et al. 2008. J. Immunol. 181:2855. PubMed
16. Fallarino F, et al. 2009. J. Exp Med. 206:2511. PubMed
17. Banham Alison, et al. 2009. Vet Immunol and Immunop 127.3-4:376-381
18. Klunker S, et al. 2009. J. Exp Med. PubMed
19. Haque A, et al. 2010. J. Immunol. 184:2583. PubMed
20. Liu Y, et al. 2013. J Mol Cell Biol. PubMed
21. Murakami R, et al. 2013. PLoS One. 8:73270. PubMed
22. Xie L, et al. 2014. J Immunol. 192:6009. PubMed
23. Prado-Garcia H, et al. 2015. Anticancer Res.35:1529. PubMed

C57BL/6 splenocytes were surface stained

C57BL/6 splenocytes were surface stained with CD4 APC and then treated with True-Nuclear™ Transcription Factor Buffer Set. Cells were then stained with FOXP3 (clone 150D) Alexa Fluor® 488 (top) or mouse IgG1, κ Alexa Fluor® 488 isotype control (bottom).





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 150D monoclonal antibody reacts with human, mouse and rat FOXP3. The 150D antibody recognizes FOXP3 epitope encoded by exon 2.

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.


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