Isolation and Stimulation of CD4+CD25+ Tregs

Peripheral blood CD4⁺CD25⁺ Tregs were collected and isolated from one healthy volunteer by magnetic cell separation using CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to our previous descriptions [14 (link)]. Isolated CD4+CD25+ Tregs were cultured and stimulated based on previously reported protocols with minor modifications [16 (link), 17 (link)]. Isolated CD4+CD25+ Tregs were cultured at a density of 2.5 × 10⁵ cells per well in a 24-well culture plate supplemented with soluble anti-CD3 (1 mg/ml), IL-2 (100 U/ml), and 50 μg/ml fresh oxidized low-density lipoprotein (ox-LDL) (Peking Union-Biology, Beijing, China). In inhibition experiments, Tregs were stimulated with inhibitors of DNA methyltransferases (5-aza-2′-deoxycytidine, AZA [10 μM] and epigallocatechin-3-gallate, EGCG [40 μM]) (Sigma-Aldrich, St. Louis, MO, USA) and were transduced with DNMT3b shRNA (multiplicity of infection =25), respectively. Tregs were cultured for 72 hours in an atmosphere of 5% CO2 at 37°C. The experiments were repeated three times and each was from one healthy volunteer. The healthy volunteer gave informed consent to participate in this study, which was approved by the Ethics Committee of Xi'an Jiaotong University.