Cytotoxicity assay.

<p>Cytotoxic responses were evaluated by the lactate dehydrogenase (LDH)-releasing method. Splenic T-cells from mice immunized intravenously with L1210-derived exosomes (LEX_L1210) and dendritic cells (DCs) pulsed with LEX_L1210 or phosphate-buffered saline (PBS) as a control were harvested and co-cultured with irradiated L1210 cells. At the end of the culture period, viable T-cells were separated using Ficoll-Paque centrifugation and were thereafter referred to as effector cells. The LDH assay is an enzymatic method used to colorimetrically quantify LDH released from lysed target cells, including L1210 or P388 cells, which served as the control and were mixed at different ratios with effector cells after incubation for 4 h at 37°C. The spontaneous/maximal release ratio was <20% in all experiments. Specific lysis (%) was calculated as follows: (experimental LDH release – effector cell spontaneous LDH release – target spontaneous LDH release)/(target maximum LDH release)×100. * <i>P</i><0.05 compared with the PBS, DC, and P388 groups; ** <i>P</i><0.01 compared with the PBS control group; △ <i>P</i><0.05 compared with the LEX_L1210 group; △△ <i>P</i><0.01 compared with the LEX_L1210 group. Experiments were performed in triplicate. One representative experiment is shown.