True-Nuclear™ One Step Staining Mouse Treg Flow™ Kit (FOXP3 Alexa Fluor® 488/CD25 PE/CD4 PerCP)

Product Details

Verified Reactivity

Mouse

Antibody Type

Monoclonal

Host Species

Rat

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-mouse FOXP3/CD25 PE/CD4 PerCP antibody cocktail - 25 tests
2. Alexa Fluor® 488 Rat IgG2b, 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 45-60 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-mouse FOXP3/CD25 PE/CD4 PerCP antibody cocktail or 20 µL of Alexa Fluor® 488 rat IgG2b, ? 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).

Application References

(PubMed link indicates BioLegend citation)

1. Ono M, et al. 2007. Nature 446:685.
2. Hori S, et al. 2003. Science 299:1057.
3. Fontenot JD, et al. 2003 Nature Immunol 4:330.
4. Fallarino F, et al. 2009. J. Immunol. 183:6033. PubMed
5. Barber A, et al. 2009 J. Immunol. 183:6939. PubMed
6. Nakashima H, et al. 2010. J. Immunol. 184:4637. PubMed

Product Citations

1. Peng K, et al. 2017. PLoS One.. 10.1371/journal.pone.0183271. PubMed
2. Lu Y, et al. 2018. Cancer Cell. 33:1048. PubMed

Antigen Details

Structure

50-55 kd protein. Forkhead/winged-helix transcription factor family, contains zinc finger and forkhead domains.

Distribution

Nuclear; expressed in Treg cells.

Function

Master regulatory gene in Treg cell development, crucial for immune homeostasis.

Interaction

Interacts with DNA

Cell Type

Tregs

Biology Area

Immunology, Transcription Factors

Molecular Family

CD Molecules, Nuclear Markers

Antigen References

1. Ono M, et al. 2007. Nature 446:685.
2. Hori S, et al. 2003. Science 299:1057.
3. Fontenot JD, et al. 2003 Nature Immunol 4:330.

Regulation

Present at high level in T reg cells. Induced by T cell activation.

Gene ID

20371 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

How stable is PerCP/Cy5.5 tandem as compared to PerCP alone?

PerCP/Cy5.5 is quite photostable and also better than PerCP alone in withstanding fixation.

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