True-Nuclear™ One Step Human Treg Flow Kit

Product Details

Verified Reactivity

Human

Reported Reactivity

Baboon, Cynomolgus, Rhesus, Pigtailed Macaque

Antibody Type

Monoclonal

Host Species

Mouse

Immunogen

Full-length FOXP3 protein

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

Recommended Usage

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. True-Nuclear™ Transcription Buffer Set - 120 tests (Cat. No. 424401)

Materials not included:
1. Cell Staining Buffer (Cat. No. 420201)
2. Single Color Compensation Controls

Immunofluorescence Staining Procedures:

1. Aliquot 100 µL of target cells to each tube.
2. Add 1 mL of the Transcription Factor 1X Fix solution to each tube, vortex, and incubate at room temperature in the dark for 30-60 minutes.
3. Without washing, add 2 mL of the Transcription Factor 1X Perm Buffer to each tube.
4. Centifuge tubes at 400 x g at room temperature for five minutes, and discard the supernatant.
5. 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. Resuspend the cell pellet in 100 µL of the Transcription Factor 1X Perm Buffer.
8. Add 20 µL of Alexa Fluor® 488 anti-human FOXP3/CD25 PE/CD4 PerCP antibody cocktail or 20 µL of Alexa Fluor® 488 mouse IgG1, κ isotype control/CD25 PE/CD4 PerCP antibody cocktail into the appropriate tubes. Incubate in the dark at room temperature for at least 30 minutes.
9. Add 2 mL of the Transcription Factor 1X Perm Buffer to each tube.
10. Centrifuge tubes at 400 x g at room temperature for five minutes, and discard the supernatant.
11. Add 2 mL of the cell staining buffer.
12. Centrifuge tubes at 400 x g at room temperature for five minutes, and discard the supernatant.
13. 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) and the Nuclear Factor Fixation and Permeabilization Buffer Set (Cat. No. 422601).

Application Notes

Additional reported applications (for the relevant formats) include: immunohistochemical staining of acetone-fixed frozen sections¹ and formalin-fixed paraffin-embedded sections^{1, 8, 19-20}, and Western blotting¹. The binding of 206D to FOXP3 can be partially blocked by 259D, but 206D does not show significant blocking effect on 259D binding.

NOTE: For flow cytometric staining with this clone, True-Nuclear™ Transcription Factor Buffer Set (Cat. No. 424401) offers improved staining and is highly recommended.

Application References

(PubMed link indicates BioLegend citation)

1. Roncador G, et al. 2005. Eur. J. Immunol. 35:1681. (IHC)
2. Yang ZZ, et al. 2006. Blood 107:3639.
3. Liu W, et al. 2006. J. Exp. Med. 203:1701.PubMed
4. Bollyky PL, et al. 2007. J. Immunol. 179:744.
5. Bell MP, et al. 2007. J. Immunol. 179:1893.
6. Tran DQ, et al. 2007. Blood doi:10.1182/blood-2007-06-094656. PubMed
7. Gao Q,et al.2007.J Clin Oncol.25:2586.(IHC) PubMed
8. Pillai V,et al. 2008. Blood 111:463.PubMed
9. Zheng Y, et al. 2008. J. Immunol. 181:1683. PubMed
10. Zonios DI, et al. 2008. Blood112:287. PubMed
11. Kavanagh B, et al. 2008. Blood. PubMed
12. Nevala WK, et al. 2009. Clin Cancer Res. 15:1931. PubMed
13. Grant J, et al. 2009. Cytometry B Clin Cytom. 76:69. PubMed
14. Nigam P, et al. 2010. J. Immunol. 184:1690. PubMed
15. Kmieciak M, et al. 2009. J. Transl. Med. 7:89. (ICFC) PubMed
16. Hartigan-O'Connor DJ,et al. 2007. J Exp Med.204:2679. PubMed
17. Raghaven S, et al. 2009. Ann Rheum Dis. 68:1908. PubMed
18. Hodi FS, et al. 2014. Cancer Immunol Res. 2:632.(IHC) PubMed
19. Sziros E, et al. 2015. Clin Cancer Res. 21:2840.(IHC) PubMed

Product Citations

1. Park S, et al. 2016. Proc Natl Acad Sci U S A. 113(52):E8379-E8386. PubMed

RRID

AB_3097006 (BioLegend Cat. No. 320127)

Antigen Details

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

Interaction

Interacts with DNA

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. Gandhi R, et al. 2010. Nat. Immunol. 11:846.

Regulation

FOXP3 is present at high levels in T regulatory cells, it can also be induced by T cell activation.

Gene ID

50943 View all products for this Gene ID 50943 View all products for this Gene ID 20371 View all products for this Gene ID 317382 View all products for this Gene ID 916 View all products for this Gene ID

UniProt

View information about FOXP3 on UniProt.org

Other Formats