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Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis. For immunofluorescent staining, the suggested use of this reagent is 5 µl per million cells or 5 µl per 100 µl of whole blood. It is recommended that the reagent be titrated for optimal performance for each application.
* Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633 nm / 635 nm. ** Alexa Fluor® 647 is a registered trademark of Molecular Probes, Inc. Alexa Fluor® 647 dye antibody conjugates are sold under license from Molecular Probes, Inc. for research use only, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents.
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 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 Biolegend's FOXP3 Fix/Perm solution to each tube, resuspend the cells (gentle 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.
* Alexa Fluor® 647 has a maximum emission of 668nm when it is excited at 633/635 nm. ** Alexa Fluor® is a registered trademark of Molecular Probes, Inc. Alexa Fluor® dye antibody conjugates are sold under license from Molecular Probes, Inc. for research use only, except for use in combination with microarrays and high content screening, and are covered by pending and issued patents.
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
C57BL/6 splenocytes surface stained with CD4 FITC, then intracellular stained with Alexa Fluor® 647 FOXP3 (150D) Flow Kit. Quadrant markers were set based on staining with Alexa Fluor® 647 mouse IgG1, κ isotype control
FOXP3 is a 50 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.
BioLegend's Alexa Fluor® 647 anti-mouse/rat/human FOXP3 Flow Kit is designed and formulated specifically for immunofluroscence staining and flow cytometric analysis of mouse/rat/human FOXP3+ Treg cells in a mixed lymphocyte population. This kit is composed of Alexa Fluor® 647 conjugated anti-mouse/rat/human FOXP3 antibody and Alexa Fluor® 647 conjugated matching isotype control and the critical buffers. It is easy to use for identification of FOXP3+ Treg cells.
Other Names:
Forkhead box protein P3, Scurfin, JM2, IPEX, Zinc finger protein JM2, T regulatory cells, Regulatory T cells
Distribution:
T regulatory cells
Antigen References:
1. Hori S, et al. 2003. Science 299:1057. 2. Fontenot JD, et al. 2003. Nature Immunol. 4:330. 3. Ferguson PJ, et al. 2000. Am. J. Med. Genet. 90:390. 4. Bennett CL, et al. 2001. Nature Genet. 27:20. 5. Allan SE, et al. 2005. J. Clin. Invest. 115:3276.
*These products may be covered by one or more Limited Use Label Licenses (see the BioLegend Catalog or our website, www.biolegend.com/terms). BioLegend products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of BioLegend. By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. Unless otherwise indicated, these products are for research use only and are not intended for human or animal diagnostic, therapeutic or commercial use.
This data display is provided for general comparisons between formats. Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors. If you need assistance with selecting the best format contact our expert technical support team.
Purified anti-mouse/rat/human FOXP3
Cell extract from HEK293T cells transfected with either human FoxP3 cDNA (Lane 1), mouse FoxP3 cDNA (Lane 2) was resolved by electrophoresis, transferred to nitrocellulose, and probed with monoclonal anti-FoxP3 antibody (clone 150D). Proteins were visualized using a goat anti-mouse secondary conjugated to HRP and a chemiluminescence detection system.
PE anti-mouse/rat/human FOXP3
C57BL/6 mouse splenocytes surface stained with CD4 FITC and then intracellular stained with PE anti-mouse/rat/human FOXP3 (clone 150D). The quadrant markers were set based on PE mIgG1, κ isotype control
Alexa Fluor® 488 anti-mouse/rat/human FOXP3
CD4+CD25+ (top) or CD4+CD25- (bottom) C57BL/6 mouse splenocytes stained with 150D Alexa Fluor® 488 (red line) and mouse IgG1,k Alexa Fluor® 488 isotype control (green line)
Alexa Fluor® 647 anti-mouse/rat/human FOXP3
C57BL/6 splenocytes surface stained with CD4 FITC, then intracellular stained with 150D Alexa Fluor® 647. Quadrant markers were set based on staining with Alexa Fluor® 647 mouse IgG1, κ isotype control
BALB/c mouse (top) or Lou rat (bottom) splenocytes surface stained with anti-mouse CD4 APC or anti-rat CD4 PE, respectively and then intracellular stained with Alexa Fluor® 488 anti-mouse/rat/human FOXP3 Flow Kit. The quadrant markers were set based on Alexa Fluor® 488 mIgG1, κ isotype control.
C57BL/6 mouse splenocytes surface stained with CD4 FITC and then intracellular stained with PE anti-mouse/rat/human FOXP3 Flow Kit™ (clone 150D). The quadrant markers were set based on PE mIgG1, κ isotype control
C57BL/6 splenocytes surface stained with CD4 FITC, then intracellular stained with Alexa Fluor® 647 FOXP3 (150D) Flow Kit. Quadrant markers were set based on staining with Alexa Fluor® 647 mouse IgG1, κ isotype control
Human Treg Flow™ Kit (FOXP3 Alexa Fluor® 488/CD4 PE-Cy5/CD25 PE)
Human peripheral blood lymphocytes stained with Human Treg Flow™ Kit (FOXP3 Alexa Fluor® 488/CD4 PE-Cy5/CD25 PE)
