Invitrogen™ eBioscience™ Mouse Regulatory T Cell Staining Kit #1

Description

Kit Contents:

• Anti-Mouse CD4 FITC (RM4-5): 50μg—Use at 0.125μg/test
• Anti-Mouse CD25 APC (PC61.5): 50μg—Use at 0.06μg/test
• Anti-Mouse/Rat Foxp3 PE (FJK-16s): 25μg—Use at 0.5μg/test
• Rat IgG2a Isotype Control PE: 50μg—Use at 0.5μg/test
• Anti-Mouse CD16/32 (Fc Block) Purified: 50μg—Use at 1-5μg/test
• Flow Cytometry Staining Buffer
• Fixation/Permeabilization Concentrate: 30mL
• Not included: Isotype controls for anti-CD4 (rat IgG2a, cat. 11-4321) and anti-CD25 (rat IgG1, cat. 17-4301)

The FJK-16s antibody reacts with mouse/rat Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3 a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Immunoblotting with FJK-16s antibody has mapped the epitope to amino acids 75-125 of the mouse Foxp3 protein. In the human, this region has been shown to be alternatively spliced at the mRNA level. Both the alternatively-spliced and non-spliced isoforms are present in the CD4+CD25+ subset of lymphocytes. Preliminary RT-PCR experiments have not revealed this alternatively-spliced isoform in mouse splenocytes, suggesting different gene regulation in the mouse and human.

Intracellular staining of mouse splenocytes with FJK-16s using the mouse Foxp3 staining sets and protocol reveals approximately 2% of total splenocytes in the C57Bl/6 strain and approximately 3-5% in the BALB/c mouse strain. Multicolor flow cytometric analysis demonstrates approximately 90% of the CD4+CD25+ cells and 4% of the CD4+CD25- cells staining with FJK-16s. B220+, CD11b+, CD11c+, and Ly6G/Gr-1+ cells do not show significant co-staining with FJK-16s. These data are consistent with a recent report which follows expression of Foxp3 using a GFP knock-in (Fontenot et al, 2005).

FJK-16s cross-reacts with rat Foxp3 This has been demonstrated by intracellular staining of Foxp3 and flow cytometry of rat splenocytes using the same method and reagents as used for mouse tissue. Please note that the CD4 and CD25 antibodies included in this kit only recognize the mouse antigens. For staining rat tissue, please use (CD4 FITC cat. 11-0040 and CD25 APC cat. 17-0390)