Fig. 3

a The 96-well plate template for experimental design. Thirty wells are required for testing one MSC batch, of which 12 wells serve as controls (the first two lines of the plate). Each additional MSC batch will require another 18 wells, which can be aligned as triplicate columns after the first batch. A total of four different batches can be analyzed in one 96-well plate. b Flow cytometry analysis of the T cell population in the negative and positive controls, at the end of 3-day co-culture experiment. The gating strategy consists of removal of debris, doublets, and dead cells and analysis of the viable cells on the CFSE histogram. Multiple fluorescent peaks are distinguished in the positive control (activated T cells), as compared to the single peak in the negative control (not activated T cells). c Phase-contrast microscopy images of irradiated MSC, resting T cells and activated T cells in culture (in the presence or absence of MSC). Note the integrity of the confluent layer of irradiated MSC after 4 days in culture (MSC only) and the lower number of T cells in the presence of irradiated MSC (MSC +T cells + beads vs. T cells + beads)