Cd4 cd25 treg cells isolation kit

The following mAbs were purchased from BD Biosciences PharMingen (San Diego, CA): FITC-labeled rat anti-mouse CD25 mAb (7D4; IgM), Fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 mAb (RM4-5; rat IgG2a), FITC-labeled anti-mouse CD8 mAb (53–6.7; rat IgG2a), phycoerythrin (PE)-labeled rat anti-mouse CD4 mAb(clone GK1.5), PE-labeled anti-mouse CD25 mAb, and PE-labeled anti-mouse CD8α mAb (53–6.7; rat IgG2a). In addition, PE-labeled anti-mouse Foxp3 mAb (FJK-16s) and its staining kit were obtained from eBiosciences (San Diego, CA).
The culture medium used in the present study was RPMI 1640 (Hyclone, Logan, UT) supplemented with 10% heat-inactivated FCS (Hyclone, Logan, UT), 100U/ml penicillin, 100μg/ml streptomycin, 2mM L-glutamine, 10mM HEPES, 1mM sodium pyruvate and 50μM 2-ME (Sigma, St. Louis, MO). DMSO was obtained from Promega Co, Ltd (USA). BSA was purchased from Zhongshan Biotec Co, Ltd (Beijing, China).CD4⁺CD25⁺Treg cells isolation kit was purchased from Miltenyi Biotec(Bergisch-Gladbach, Germany). Mitomycin C (C15H18N4O5) was obtained from Jinmei Co, Ltd. (Beijing, China). [³H] thymidine was purchased from China institute of atomic energy (Beijing, China).

Dead cells were removed by dead cell removal kit (Miltenyi Biotec, Auburn, California, USA, Cat# 130-090-101). CD4⁺CD25^hiFoxp3⁺Treg cells and CD4⁺CD25⁻Teffs were isolated from PBMC using the CD4⁺CD25⁺Treg cells isolation kit (Miltenyi Biotec, Cat# 130-091-301) according to manufacturer's instruction (Supplementary 4). The purification of Tregs and Teffs were determined by FACS Aria II flow cytometer (BD biosciences, San Jose, California, USA) using CD4 APC (Biolegend, San Diego, California, USA, Cat# 300514) and CD25 PE (Biolegend, Cat# 302605).
Before culture, 24-well round-bottom plates were preincubated with anti-CD3 (10 μg/ml; Biolegend, Cat# 300414) at 37°C for 2 hours. Teffs were cultured with Tregs at 8:1 ratio or without Tregs. The cells were cultured in 1 ml per well in 24-well round-bottom plates which were preincubated and stimulated with anti-CD28 (2.5 μg/ml, Biolegend, Cat# 302914) and 10 ng/ml interleukin-2 (IL-2; Biolegend, Cat# 589102).

CD4⁺CD25⁻ T and CD4⁺CD25⁺ Treg cells from thymic glands, PDLNS and spleen were sorted by using the Miltenyi Biotec magnetic sorter (Miltenyi Biotec, Germeny) and the CD4⁺CD25⁺ Treg cells isolation kit (Miltenyi Biotec) as described previously32 (link).