Cytotox 96 nonradioactive ldh assay

The LDH procedure was performed as described elsewhere (21 ). YAC-1 cells are murine lymphoma cells that are sensitive to cytotoxic immune cells, including CD3⁺CD8⁺ T and NK cells. Co-culture of the splenocytes with YAC-1 cells has been commonly used to evaluate the cytotoxicity of CD8 T and NK cells (24 (link)). The cytotoxicity activity of coconut juice vinegar was measured using a Cytotox 96 nonradioactive cytotoxicity assay kit (Promega, Wisconsin, USA). In brief, the spleen was harvested from the untreated or coconut water vinegar-treated mice, and the splenocytes were isolated and incubated with YAC-1 cells for 24 h. The ratios of splenocytes (effector spontaneous) to YAC-1 cells (target spontaneous) used in this study were 2:1 and 5:1. The cytotoxicity of the splenocytes on YAC-1 cells was tested by the Cytotox 96 nonradioactive LDH assay (Promega, Wisconsin, USA) according to the manufacturer’s protocol.

BMdM were primed with 20 ng/mL lipopolysaccharide (Sigma) for NLRP3 inflammasome activation and treated with 100-nm DOPG liposomes (50 μM) for 1 h before silica (50 μg/mL) application. BMdM were incubated (37°C; 5% CO₂) for 24 h in 96 well plate (100,000 cells/well) in complete RPMI 1640 media (10% fetal bovine serum, 1% Penicillin/Streptomycin). Cell death was measured in supernatants by Promega CytoTox 96 Non-Radioactive LDH assay. IL-1β release was quantified in supernatants by R&D Systems IL-1β ELISA kit.