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)