Quantifying Cell Death by LDH Assay

LDH release into cell culture medium was used as an indicator of cell death using NAD⁺ reduction assay (Roche Applied Science). Monocytes were grown in culture tubes at the density 10⁶/ml and pretreated with or without inhibitor for 30 minutes followed by LPS (1μg/ml) for 0.5 or 3 hours and finally activated with 5mM ATP for 30 minutes. Cell culture medium was collected, clarified by centrifugation and used for LDH assay. Total LDH content in cells (positive control) was measured in cells lysed with Triton X-100 (1% final concentration). Cell culture medium alone was used as a blank and OD values were subtracted from readings of samples and positive control. LDH concentration in the medium was detected at wavelength 490 nm. Cell death was calculated by the formula:
$\text{cytotoxicity}(\%)=[(\text{sample}-\text{blank})/(\text{positive}\text{control}-\text{blank})\times 100]$ , as described earlier (30 (link)).