Cytotoxicity analysis using carboxyfluorescein diacetate succinimidyl estercarboxyfluorescein diacetate succinimidyl ester (CFSE)-propidium iodide (PI) staining-based Flow cytometry. CFSE- and PI-positive target cells represent cells lysed by effector cells. (A) Spleen cells from tumor-bearing mice killed fewer slow-cycling and drug-resistant 5-fluorouracil (FU)-treated CT-26 tumor cells. No significant differences were seen (t-test). (B) Spleen cells from tumor-bearing mice immunized with inactivated FU-CT-26 or non-FU-CT-26 cells showed tumor-specific cytotoxicity in vitro, and spleen cells from mice immunized with inactivated FU-CT-26 cells exhibited higher cytotoxicity compared with the other two groups. (*P < 0.05, **P < 0.01, t-test) Error bars represent the standard deviation. From left to right, the target cells respectively were FU-CT-26, non-FU-CT-26, 4T-1, and YAC-1 cells. Effector:target cell ratio was 50:1. SP/FU-CT-26: Spleen cells from tumor-bearing mice immunized with mitomycin C (MMC)-inactivated FU-CT-26 cells. SP/non-FU-CT-26: Spleen cells from tumor-bearing mice immunized with MMC-inactivated CT-26 cells. SP/Control: Spleen cells from tumor-bearing mice treated with PBS. Experiments were repeated three times with similar results.