Cd4 cd25 t cell isolation kit

Mouse spleens were meshed through a cell strainer into Phosphate Buffered Saline (PBS) with 2% Fetal Bovine Serum and 1% Penicillin/Streptomycin, and cells were collected by spinning at 1000 rpm for 10 min. CD4+CD25- T cells were purified by using CD4+CD25+ T cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, cells were incubated with Biotin-conjugated antibodies, followed by anti-biotin microbeads and PE-conjugated anti-CD25 antibody for 30 min on ice with gentle shaking. Mixtures were loaded into pre-washed LD columns in the Magnetic field, and CD4+ cells were collected and then incubated with anti-PE microbeads. Mixtures were loaded into prewashed MS columns in the magnetic field to collect CD4+CD25- T cells (non-bound cell fraction). Cells were plated into 12-well plates pre-coated with 1μg/ml anti-CD3e (eBioscience) overnight in RPMI1640 Medium with 10% FBS, 1% Penicillin/Streptomycin, 1% non-essential amino acids, and 2-mercaptoethanol, 1µg/ml anti-CD28 (BD Biosciences), and 1ng/ml TGF-β1 (Peprotech). Cells were cultured at 37°C up to five days for the conversion of CD4+CD25- T cells to CD4+CD25+ T regs. For flow cytometry, cells were stained with anti-mouse CD25 Alexa Fluo®488 (eBioscience) and analyzed by using multicolor FACS (ARIA; BD Biosciences) as previously described.^{12 (link)}