True-Nuclear™ Human Treg Flow Kit

Product Details

Verified Reactivity

Human

Antibody Type

Monoclonal

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-mouse/rat/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-mouse/rat/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 over the Foxp3/Perm Buffer Set (Cat. No. 421403).

Application References

(PubMed link indicates BioLegend citation)

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. 2012. Food Chem Toxicol. 50:1920. PubMed

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RRID

AB_3097007 (BioLegend Cat. No. 320027)

Antigen Details

Distribution

T regulatory cells

Cell Type

Tregs

Biology Area

Cell Biology, Immunology, Transcription Factors

Molecular Family

CD Molecules, Nuclear Markers

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.

Gene ID

20371 View all products for this Gene ID 317382 View all products for this Gene ID 50943 View all products for this Gene ID

UniProt

View information about FOXP3 on UniProt.org

Other Formats