2.12. In vivo cytotoxicity assay

C57BL/6 mice were immunized with PBS, 2.5 mg/kg OVA, 50 mg/kg CFP, or a combination of OVA and CFP on day 7 and 14. Five days after the last treatment, target cells were transferred into the immunized mice. The splenocytes from naïve mice were extracted and 10 × 10⁶ splenocytes were labeled with 10 mM Cell Tracker Orange CMTMR (Life Technologies, Carlsbad, California, US; #M7510) and loaded with control peptide. Another 10 × 10⁶ splenocytes were labeled with 200 nM CFSE (Invitrogen, San Diego, California, US; #{"type":"entrez-nucleotide","attrs":{"text":"C34554","term_id":"2370695","term_text":"C34554"}}C34554) and 100 nM SIINFEKL peptide was loaded. A mixture containing each target cell population (a total of 10 × 10⁶ cells) was intravenously injected to immunized mice. Percentage killing was calculated using the formula described by FACS Fortessa (Becton Dickinson, Franklin Lakes, New Jersey, US).