Materials Provided:

1. Alexa Fluor® 488 anti-human FOXP3/CD25 PE/CD4 PerCP antibody cocktail, 25 tests
2. Alexa Fluor® 488 Mouse IgG1, κ isotype control/CD25 PE/CD4 PerCP antibody cocktail, 25 tests
3. FOXP3 Fix/Perm buffer and FOXP3 Perm buffer , 100 tests Cat. No. 421403

Materials not included:
Cell Staining Buffer (Cat. No. 420201)

Immunofluorescence Staining Procedures:

Centrifugation steps: perform at 250Xg for 5min
Incubation steps: perform at room temperature.

1. Prepare 1X working solutions of FOXP3 Fix/Perm buffer and FOXP3 Perm buffer prior to staining. 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 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.

2. Suspend the cells of interest at 1X10⁷/ml  in BioLegend's cell staining buffer (Cat. No. 420201).
3. Distribute 100 μl of the cell suspension into each FACS tube.
4. Add 1 ml of 1X BioLegend's FOXP3 Fix/Perm solution to each tube, vortex and incubate for 20 minutes; centrifuge, then discard the supernatant.
5. Wash: resuspend cells in 2ml/tube cell staining buffer; centrifuge, then discard the supernatant.
6. Wash: resuspend in 1ml 1X BioLegend's FOXP3 Perm buffer; centrifuge, then discard the supernatent.
7. Resuspend cells in 1ml 1X BioLegend's FOXP3 Perm buffer, incubate in the dark for 15 minutes; centrifuge, then discard the supernatant.
8. Add 20 μl Alexa Fluor® 488 anti-human FOXP3/CD25 PE/CD4 PerCP antibody cocktail or 20 μl Alexa Fluor® 488 mouse IgG1, κ isotype control/CD25 PE/CD4 PerCP antibody cocktail into appropriate tube, and resuspend the cell pellet by gentle vortex. Incubate in the dark for 30 minutes.
9. Wash twice with cell staining buffer (see step 5) and resuspend in 0.5ml cell staining buffer then analyze with flow cytometer using appropriate instrument settings.

1. Roncador G, et al. 2005 Eur. J. Immunol. 35:1681.
2. Yang ZZ, et al. 2006. Blood 107:3639. PubMed
3. Liu W, et al. 2006. J. Exp. Med. 203:1701.PubMed
4. Groh V, et al. 2006. Nature Immunology 7:755. PubMed
5. Lewkowicz P, et al. 2006 J. Immunol. 177:7155. PubMed
6. Bollyky PL, et al. 2007. J. Immunol. 179:744.
7. Bell MP, et al. 2007. J. Immunol. 179:1893.
8. Tran DQ, et al. 2007. Blood doi:10.1182/blood-2007-06-094656. PubMed
9. Gao Q,et al.2007.J Clin Oncol.25:2586.PubMed
10. Pillai V,et al. 2008. Blood 111:463.PubMed
11. Zheng Y, et al. 2008. J. Immunol. 181:1683. PubMed
12. Zonios DI, et al. 2008.Blood112:287. PubMed
13. Kavanagh B, et al. 2008. BloodPubMed
14. Nevala WK, et al. 2009. Clin Cancer Res. 15:1931. PubMed
15. Grant J, et al. 2009. Cytometry B Clin Cytom. 76:69. PubMed
16. Nigam P, et al. 2010. J. Immunol. 184:1690. PubMed
17. Kmieciak M, et al. 2009. J. Transl. Med. 7:89. (ICFC) PubMed
18. Hartigan-O'Connor DJ,et al.2007.J Exp Med.204:2679. PubMed
19. Raghaven S, et al. 2009. Ann Rheum Dis. 68:1908. PubMed