Anti human cd3 and cd28 antibodies

Unprimed or cytokine-primed hUC-MSCs (1 × 10⁵ cells) were plated in a 24-well plate (Corning) and cultured for 12 h before they were used for the lymphocyte proliferation assay. Human PBMCs were washed twice with D-PBS and stained with CFSE (5 µmol/l; Invitrogen), which was used to assess T-cell proliferation. The cells were then suspended in RPMI 1640 at 1 × 10⁶ cells/ml and distributed into 24-well plates (1 ml/well) in the presence or absence of hUC-MSCs. To induce T-cell proliferation, anti-human CD3 and CD28 antibodies (BD Pharmingen; final concentration, 500 ng/ml) were added to the wells. After four days of coculture, the CD3⁺ T cells were collected and analysed via flow cytometry. Immunosuppression rate (%) = [(A-B)/A] × 100%, where A is the proliferation rate of T cells without MSC coculture (positive group) and B is the proliferation rate of T cells with cytokine-primed MSC coculture (experimental group).

Human hUC-MSCs (1 × 10⁵ cells) were plated to a 24-well plate (Corning) and cultured for 24 h they were used for the lymphocyte proliferation assay. Human PBMCs were washed twice with D-PBS and stained with CFSE (5 μmol/l, Invitrogen), which was used to assess T-cell proliferation. The cells were then suspended in Roswell Park Memorial Institute (RPMI)1640 at 1 × 10⁶ cells/ml and distributed to 24 well plates (1 ml/well) in the presence or absence of hUC-MSCs. To induce T-cell proliferation, anti-human CD3 and CD28 antibodies (BD Pharmingen; final concentration, 500 ng/ml) were added to the wells. After four days of coculture, the CD3⁺ T-cells were collected and analyzed by flow cytometry.