Fitc conjugated anti mouse cd4 clone gk1.5

Flow cytometric detection of regulatory T cells was performed using fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (clone GK1.5; eBioscience, San Diego, CA, USA), phycoerythrin (PE)-conjugated anti-mouse CD25 (clone PC61.5; eBioscience, San Diego, CA, USA), and Alexa Fluor 647 anti-mouse Foxp3 (clone MF23; BD Pharmingen™, San Jose, CA, USA). The single-cell splenocytes were washed with PBS and stained with FITC-conjugated anti-CD4 and PE-labeled anti-CD25 antibodies in staining buffer. The cells were subsequently fixed and stained with Alexa Fluor 647 anti-Foxp3 antibody overnight at 4°C in the dark. After washing, the cells were stored at 4°C in the dark for subsequent detection.

Recombinant mouse cytokines were obtained from R&D Systems. Propidium Iodide (PI) was obtained from Sigma-Aldrich. FITC-conjugated anti-mouse CD4 (clone GK1.5), APC-conjugated anti-CD8 (Ly-2) and PE-conjugated anti-CD25 (PC61) mAbs were purchased from eBioscience. Purified anti-CD3ε (145-2C11), anti-CD28 (37.51), anti-mouse-IL-2(S4-B6) and anti-mouse Bcl2 staining kit were purchased from BD Biosciences. Mitotracker Red staining kit to measure mitochondrial membrane potential and anti-CD3/CD28 coated beads were purchased from Molecular Probes (Invitrogen).