In vivo cytotoxicity killing assay

C57BL/6 mice were injected subcutaneously with 10 µg of OVA with or without UNE-C1 once a week. After 7 days of second injections, splenocytes from naïve C57BL/6 mice were harvested. Half of the splenocytes were pulsed with SIINFEKL peptide, and half remained unpulsed. After 2 hours, pulsed and unpulsed groups were labeled with 2.5 and 0.25 µM of carboxyfluorescein succinimidyl ester (CFSE, Invitrogen), respectively, for 15 min and the remaining dyes were quenched with serum media. Equal numbers of pulsed and unpulsed cells were mixed evenly and injected intravenously into the immunized mice. After 24 hours, splenocytes were collected, and CFSE^high and CFSE^low cells were determined and specific target cell killing was calculated with the following formula: [1–(% of CFSE^high in immunized sample/% of CFSE^low in immunized sample)/(% of CFSE^high in unimmunized sample/% of CFSE^low in unimmunized sample)]×100.