Anti cd3 cd28 mab coated dynabeads

Human peripheral blood (PB) leukapheresis products of volunteers were obtained from the Department of Hematology in the Affiliated Jiangning Hospital of Nanjing Medical University. Naive human PB T cells (CD4⁺CD45RA⁺) were sort purified from PB mononuclear cells (PBMCs) (Ficoll-Hypaque, Amersham Biosciences) by magnetic-activated cell sorting (MACS pro) (Miltenyi Biotec, Germany) in a two-step procedure of magnetic beads sorting.
Naive T cells were induced to iTregs with anti-CD3/CD28 mAb-coated Dynabeads (Life Technologies, Carlsbad, CA, USA) at 1:1 (cell-to-bead) ratios in the presence of tumor growth factor-β (TGF-β) (1 ng/ml)(Bio-Techne, Abingdon, OX, USA) and recombinant interleukin-2 (IL-2) (100 U/ml) (Chiron, Emeryville, CA, USA) in X-Vivo-15 (BioWhittaker, Walkersville, MD, USA) media supplemented with 10% fetal bovine serum (Valley Biomedical) for 72 h.
The iTregs were cultured in the same media with the addition of recombinant IL-2 (300 U/ml) at the concentration of 0.5 × 10⁶ cells/ml. IL-2 (300 U/ml) was added every 2 or 3 days. iTregs were treated with miR inhibitor or miR mimic (Ribobio Corporation, Guangzhou, China) and renewed together with IL-2 on point days. The inhibitor group was treated with miR-142-3p inhibitor (100 nM), while the mimic group was treated with miR-142-3p mimic (50 nM). Cells were collected and tested as described.

Peripheral blood (PB) leukapheresis products were obtained from volunteers in Nanjing Medical University. Naïve human PB tTreg (CD4⁺CD25⁺CD127⁻) were sort-purified from PB mononuclear cells (PBMNCs) (Ficoll-Hypaque, Amersham Biosciences) in a two-step procedure.
tTreg cells were stimulated with anti-CD3/CD28 mAb-coated Dynabeads (Life Technologies, Carlsbad, CA) at 1:3 (cell to bead) ratios in the presence of recombinant IL-2 (300 U/ml) (Chiron, Emeryville, CA) in X-Vivo-15 (BioWhittaker, Walkersville, MD) media supplemented with 10% human AB serum (Valley Biomedical) on day 0. Cells were counted and cultured at the concentration of 0.5 × 10⁶ cells/ml and IL-2 (300 U/ml) was renewed every 2 or 3 days. On point days (day 0 or 14), cells were re-suspended at 0.5 × 10⁶ cells/ml and treated with antagomir or agomir and renewed together with IL-2. Cells were harvested and assayed as listed.