One Step Staining Mouse Treg F

FC, ICFC - Quality tested

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. FOXP3 Fix/Perm Buffer Set, 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-mouse FOXP3/CD25 PE/CD4 PerCP antibody cocktail or 20 µl Alexa Fluor® 488 rat IgG2b, 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. Ono M, et al.:Nature 2007 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

T regulatory (Treg) cells are a subset of T lymphocytes which is characterized by CD4⁺/CD25⁺/FOXP3⁺. These naturally occurring Treg cells originate in the thymus, and comprise 2-10% of peripheral CD4⁺ T cells. It has been shown that Treg cells are able to inhibit T cells proliferation and cytokine production and play critical roles in preventing autoimmunity as well as in controlling tumor immunity and transplantation tolerance. Impaired Treg function or Treg cell deficiency will develop variety of autoimmune diseases, while higher frequency of Treg cells will cause hypo-immune response to pathogens.

BioLegend's One Step Staining Mouse Treg Flow™ Kit is designed and formulated specifically for immunofluorescence staining and flow cytometric analysis of mouse Treg cells in a mixed lymphocyte population. This kit is composed of fluorochrome conjugated anti-mouse CD4, CD25 and FOXP3 antibodies and the critical buffers. It is an one-step staining kit for labeling both surface and intracellular antigens of Treg cells.