Ldh cytotoxicity assay kit

For the ADCC assay, ENKTL cells (target cells) were incubated with LMP1-IgG. An unrelated IgG was used as the control. PBMCs were used (effector cells) and incubated with ENKTL cells at a fixed effector/target ratio of 25:1. After a 4-hour incubation at 37°C, the cell supernatants were added to a 96-well plate to evaluate LDH release by LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China). For the CDC assay, ENKTL cells were incubated with LMP1-IgG, and this was followed by the addition of 20% human serum or heat-inactivated human serum. Then, the cell supernatants were added to a 96-well plate to evaluate LDH release with a LDH Cytotoxicity Assay Kit (Beyotime). Both assays were performed according to the manufacturer's instructions [67 (link)].

Cytotoxicity was measured using a lactate dehydrogenase (LDH) cytotoxicity assay kit (Beyotime, Shanghai, China). Each strain was tested in triplicate. The human lung carcinoma A549 cell line was cultured in Roswell Park Memorial Institute (RPMI) medium with 10% (vol/vol) heat-inactivated fetal bovine serum at 37°C with 5% CO₂. Each well of a 24-well plate was inoculated with 2 × 10⁵ A549 cells and cultured overnight. Bacteria were cultured at 37°C in LB medium to an optical density at 600 nm (OD₆₀₀) of 1. One milliliter of the bacterial cells was collected by centrifugation at 10,000 × g for 1 min and washed with phosphate-buffered saline (PBS). The bacteria were resuspended in 1 mL PBS, resulting in 1 × 10⁹ CFU/mL. The A549 cells were washed once with PBS and infected with the bacteria at a multiplicity of infection (MOI) of 50 in RPMI without serum for 1 h. LDH in the medium was measured using an LDH cytotoxicity assay kit (Beyotime, Shanghai, China). The control for the total LDH release was the cells incubated with the LDH release buffer (provided by the kit). Calculation of the cytotoxicity percentage was performed following the manufacturer’s instructions.