Cyto tox 96 non radioactive cytotoxicity assay kit

Single-cell suspensions were extracted from the spleens of 6-week-old C57BL/6 mice to isolate CD8⁺ T-cells. CD8⁺ T cells were purified using a mouse CD8⁺ isolation kit (Stemcell, Canada). Then, the harvested CD8⁺ T cells were seeded at a density of 1×10⁵ in 96-well plates and supplemented with anti-CD28 (1 μg/mL; BioLegend), anti-CD3 (5 μg/mL; Biolegend), and IL-2 (40 U/mL) for 3 d to induce CD8⁺ T cell activation. SCC9 cells, given the different treatments (PBS, GDYO, cholesterol, GDYO + L + Cholesterol, GDYO + L), were co-cultured for 5 hours with activated T cells at various ratios of effector to target. Using a Cyto Tox 96 Non-Radioactive Cytotoxicity Assay kit (Beyotime, China), the supernatant from each well was used for a lactate dehydrogenase cytotoxicity assay to measure target cell mortality, according to the manufacturer’s recommended protocol. In other experiments, activated CD8⁺ T cells were added at the effector to target ratios of 10:1 for 5 h to quantify the death of cancer cells by flow cytometry, and then co-culture media were measured for the inflammatory mediators, including IFN-γ and TNF-α, using enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific, USA).