True-Nuclear™ Human Treg Flow™ Kit (FOXP3 Alexa Fluor® 488/CD4 PE-Cy5/CD25 PE)

Product Details

Verified Reactivity

Human

Reported Reactivity

Cynomolgus, Rhesus, Baboon, Chimpanzee

Antibody Type

Monoclonal

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

Application Notes

Materials Provided:
1. Alexa Fluor® 488 anti-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-Cy5/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-Cy5/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-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.

Application References

(PubMed link indicates BioLegend citation)

1. Roncador G, et al. 2005 Eur. J. Immunol. 35:1681.
2. Yang ZZ, et al. 2006. Blood 107:3639. PubMed
3. Gavin MA, et al. 2006. P. Natl. Acad. Sci. USA 103:6659. PubMed
4. Groh V, et al. 2006. Nature Immunology 7:755. PubMed
5. Tran DQ, et al. 2007. Blood doi:10.1182/blood-2007-06-094656.PubMEd
6. Long SA, et al. 2008. J Autoimmun. 30:293. PubMed
7. Gong G, et al. 2009. Blood 113:837. PubMed
8. Long SA, et al. 2009. Eur J. Immunol. 39:612. PubMed
9. Long SA, et al. 2010. Diabetes. 59:407. PubMed
10. Ferraro A, et al. 2014. PNAS. 111:1111. PubMed
11. Vudattu NK, et al. 2014. J Immunol. 193:587. PubMed
12. Dupont G, et al. 2014. Cytokine. 69:146. PubMed

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.

Regulation

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

Gene ID

50943 View all products for this Gene ID

UniProt

View information about FOXP3 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

What type of PE do you use in your conjugates?

We use R-PE in our conjugates.

Can I stain whole blood with anti-FOXP3 using your Foxp3 staining kit?

It is not recommended. It is best to use PBMCs for this testing.

Can FOXP3 be costained with cytokines?

The larger holes created by the nuclear permeabilization required for FOXP3 may allow cytokines to leak out of the cell, making it harder to detect lowly-expressed cytokines. You may have to use a control where the cells are only permeabilized through the cell membrane.

Other Formats

Certificate of Analysis