Lymphocyte proliferation assay

EPCs were pre-treated with IFN-γ (10 ng/ml, 48 h) or left untreated. A total of 5×10⁴ cells/well stimulator cells (pre-treated EPCs; un-treated EPCs; MSCs; pre-treated EPCs and MSCs (2:3); un-treated EPCs and MSCs (2:3)) were washed and seeded into a 96-well plate separately. For mixed lymphocyte reactions (MLR), all the attached stimulator cells were pre-treated with 10 μg/ml mitomycin C for 2 h.

Human PBMCs were collected from healthy individuals who gave informed consent. Allo-PBMCs were isolated from human peripheral blood by density gradient centrifugation using Ficoll. Isolated PBMCs were incubated with red blood cell lysis buffer (Qiagen, Germany) for 5 min at room temperature and washed twice with DPBS. A total of 2×10⁵ PBMCs were labeled with CFDA-SE (2 μM, 5 min; Beyotime), added to each well and co-cultured with different stimulator cells. All cells were cultured in RPMI 1640 (HyClone, USA) with 10% FBS and 2 μM L-glutamine (Sigma, USA). PBMCs cultured alone or stimulated by mitogen phytohaemagglutinin (PHA, 5 μg/ml; Sigma, Australia) acted as a negative control and positive control, respectively. After one week of co-culture, suspended PBMCs were harvested; proliferation of lymphocyte and T lymphocyte subsets were analyzed by flow cytometry.