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Centrifugation steps: perform at 250Xg for 5min Incubation steps: perform at room temperature.
1. Distribute 0.5-1 X 106 cells/100 μl/tube into FACS tubes 2. Add 20 μl of CD4 APC/CD25 PE cocktail to each tube, vortex and incubate in the dark for 20 minutes. 3. Wash once with cell staining buffer (Cat. No. 420201); centrifuge, then discard the supernatant. 4. 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 heavy precipitation observed after diluted to 1X working solution, it can be filtered to clarify the solution. Caution: The FOXP3 Fix/Perm buffer contains paraformaldehyde, which is toxic and mutagenic. Please wear necessary protection to avoid direct body contact.
5. 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. 6. Wash: resuspend cells in cell staining buffer (Cat. No. 420201); centrifuge, then discard the supernatant. 7. Wash: resuspend in 1ml 1X BioLegend's FOXP3 Perm buffer; centrifuge, then discard the supernatant. 8. 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. 9. Add 5 μl Alexa Fluor® 488 anti-mouse FOXP3 antibody or 5 μl Alexa Fluor® 488 mouse IgG1, k isotype control into appropriate tube and incubate in the dark for 30 minutes. 10. Wash twice with cell staining buffer (see step 6), and resuspend in 0.5ml cell staining buffer then analyze with flow cytometer using appropriate instrument settings.
* Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 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. PubMed 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
T regulatory (Treg) cells are a subset of T lymphocytes which is characterized by CD4+/CD25+/FOXP3+. These naturally occurring Treg cells originate in the thymus, and comprise 2-10% of peripheral CD4+ T cells. It has been shown that Treg cells are able to inhibit T cells proliferation and cytokine production and play critical roles in preventing autoimmunity as well as in controlling tumor immunity and transplantation tolerance. Impaired Treg function or Treg cell deficiency will develop variety of autoimmune diseases, while higher frequency of Treg cells will cause hypo-immune response to pathogens.
BioLegend's Mouse Treg Flow™ Kit is designed and formulated specifically for immunofluroscence staining and flow cytometric analysis of mouse Treg cells in a mixed lymphocyte population. This kit is composed of flurochrome conjugated anti-mouse CD4, CD25 and FOXP3 antibodies and the critical buffers. It is easy to use for identification of Treg cells.
Other Names:
Forkhead box protein P3, Scurfin, JM2, IPEX, Zinc finger protein JM2, T regulatory cells, Regulatory T cells
*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)
