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Figure 2: Cellular characterization after preactivation of MSCs. Viability test and MHC class II expression were performed 24 h and 96 h after MSC preactivation with 20 ng/mL IFN-γ and 30 ng/mL TNF-α for 20 h. The frequency of dead cells (a) in the preactivated and untreated groups was estimated by propidium iodide incubation and flow cytometry analysis (). MHC-II expression (b) was assessed by anti-MHC class II antibody (red peaks) and isotype control (grey peaks) on the preactivated and untreated MSCs after 24 and 96 h. The frequency of MHC class II-positive cells (c) from three independent experiments is presented (). Splenocyte proliferation assay was performed by coculture of splenocytes with preactivated and untreated allogeneic or syngeneic MSCs. Proliferation was measured by BrdU incorporation after 72 h coculture with the preactivated and untreated MSCs (d). The stimulation index (SI) was calculated by the ratio between the absorbance of the MSC/splenocyte coculture group and the splenocytes only (control group). Analysis of activated T cells after coculture for 72 hours with syngeneic or allogeneic mesenchymal stem cells (e and f). No statistically significant differences were found in the frequency of activated T helper cells (CD4+/CD69+) (e) or T cytotoxic cells (CD8+/CD69+) (f) when splenocytes were cocultured with syngeneic or allogeneic MSCs at 1 : 4 or 1 : 10 MSC to splenocyte rates (). Two-way ANOVA, post hoc Tukey. Allo: allogeneic MSCs; Syn: syngeneic MSCs. Significant difference between allogeneic () and syngeneic () groups. Two-way ANOVA, post hoc Tukey. .