Annexin pi assay

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).

Tris (Dibenzylideneacetone) dipalladium (Tris DBA) was prepared by Dr Arbiser as previously described [25 (link)]. Propidium iodide (PI), RNAase and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solutions were from Sigma-Aldrich (St Louis, MO), total protein concentration was assessed by Quick Start™ Bradford dye reagent (BioRad, Hercules, CA), mAbs for Western blotting from Cell Signaling Technologies (Danvers, MA), and Annexin/PI assay from BD Biosciences (San Jose, CA). Red blood cell (RBC) lysis buffer 10 × was obtained from BioLegend (San Diego, CA). Drugs including bortezomib and carfilzomib were purchased from Selleck Chemicals (Houston, TX).