2.16. LDH-Based T Cell Killing Assay (Cytotoxicity Assay)

1 × 10⁴ target cells (SMMC7721) were seeded in triplicate in a 96-well U-bottom plate with an effector (human peripheral blood mononuclear cell (PBMC)) (SailiBio, Inc.) at a ratio of 10 : 1 (effector : target (E : T)) and coincubated in 200 μL of RPMI-1640 containing 4% FBS for 6 h at 37°C with 5% CO₂. The cytotoxicity was measured by the lactate dehydrogenase (LDH) release assay (Non-Radioactive Cytotoxicity Assay, Promega). The absorbance at 490 nm was recorded by using a microplate reader (BioTek Instruments, Inc., USA). Except for wells with E+T (samples), other wells were designed for controls: wells only with a medium (blank), wells only with PBMC (E only), and wells only with target cells (T only). Each control well was triplicated. The T only wells were completely lysed by adding 30 μL lysis solution (10x). The cytotoxic rate % was calculated according to the following formula: cytotoxic rate% = (samples − E only − blank)/(T only − blank)∗100.

Cell-free supernatants from T cell and tumor cell coculture were harvested at 6 h for the presence of interleukin-2 (IL-2) and interferon-γ (IFN-γ). These cytokines were quantified by ELISA kits (Boster, China). All samples were tested in duplicate according to the manufacturer's instructions.