Immunosuppressive Capacity of Irradiated MSCs

Murine MSCs with different treatments were irradiated with 30 Gy from a 137 Cs source as previously described^{11 (link)}, to inactivate MSCs by inhibiting their proliferation while reserving their immunosuppressive capacity, and then seeded into 96-well plates. Freshly isolated splenocytes (2 × 10⁵ cells/well) from C57/BL6 mice were labeled with 7.5 μM carboxyfluorescein diacetatesuccinimidyl ester (CFSE, Thermo Fisher Scientific) and co-cultured with murine MSCs for 3 days in the presence of mouse anti-CD3/CD28 antibodies (eBiosciences, San Diego, CA, USA), then collected for flow cytometric analysis on a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Human MSCs with different treatments were irradiated with 30 Gy from a 137 Cs source and seeded into 96-well plates. Freshly isolated PBMCs (2 × 10⁵ cells/well) from healthy volunteers were labeled with 7.5 μM CFSE and co-cultured with human MSCs for 3 days in the presence of human anti-CD3/CD28 antibodies (eBiosciences), then collected for flow cytometric analysis on a FACS Calibur flow cytometer.