Nad reduction 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)).

Cell death was quantified by lactate dehydrogenase (LDH) release in cell culture medium using NAD⁺ reduction assay as per manufacturer’s protocol (Roche Applied Science) and Annexin/PI assay following manufacturer’s protocol (BD Pharmingen). IMR90 fibroblasts plated in a 48-well plate at the density 30,000 cells/well were stimulated with EVs isolated from Thp1, Cas9 or Cas9 GsdmD knockout (KO) monocytes that were untreated (CMVs) or LPS treated (LMVs) for 18 hrs. Cell culture medium was collected, clarified by centrifugation at 300 x g for 10 min, and used for detection of LDH. Cell culture medium alone was used as a blank. LDH concentration in the medium was detected at a wavelength 490 nm and data represented as the measured absorbance. Recipient fibroblasts were separated from the culture medium and stained with annexin/propidium iodide. For a positive control, fibroblasts were treated with 1 μM Staurosporine for 24 hours. Samples were analyzed by BD FACSymphony^™ A1 Cell Analyzer (BD Biosciences, San Jose, CA) and FlowJo 10 software (FlowJo, LLC, Ashland, OR).