Abstract: Objective To develop a method for evaluation of the cytotoxicity activity of mice splenic lymphocytes to human HepG2 cells based on flow cytometry(FCM), in addition to ensure the efficiency of evaluation of the cytotoxicity activity of mice splenic lymphocytes to human HepG2 cells in the process of establishing an animal model of human tumor xenotransplantation, which can preserve the biological characteristics of human tumors, meanwhile, has relatively normal immune function. Methods Human HepG2 cells were labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester) and treated with anhydrous ethanol to mimic the cytotoxic effect of cytotoxic killer cells, then determined by FCM after propidium iodide(PI) staining, based on which the concentrations of CFSE and PI and time for treatment were optimized. The lymphocytes were isolated from spleen of mice as effector cells and human HepG2 cells were target cells. A procedure was developed based on the optimization of conditions including the action time and ratio between effector and target cells. Results The cells were divided into CFSE⁺PI^-, CFSE⁺PI⁺, CFSE^-PI⁺ and CFSE^-PI^- groups by CFSE/PI staining, and the survival and killed cells were well distinguished. The concentration of CFSE was 2.5µmol/mL, the optimal action time between effector and target cells was 12 h, and the effector to target cell ratios of 10∶1 was adopted. Conclusions A method for evaluation of the killing activity of mice splenic lymphocytes to human HepG2 cells based on FCM was successfully developed, which provided a reference for evaluating the killing effect of animal splenic lymphocytes on xenogeneic cells.

Key words: Mouse, Splenic lymphocytes, Human HepG2 Cells, Carboxyfluorescein diacetate succinimidyl ester(CFSE), Propidium iodide(PI), Flow cytometry(FCM), Cytotoxicity activity