Lactate Dehydrogenase Assay

Cell death after cytokine or EV exposure was assessed using the Pierce^TM lactate dehydrogenase (LDH) Cytotoxicity Assay (Fisher Scientific) kit. Reaction substrates were prepared as per the manufacturer’s instructions. LDH assay was performed with the media from the XF^e96 cell culture microplate. Forty-five minutes prior to the end of the 24-h exposure period, 10 μl 10× lysis buffer was added to one control well, and the plate was placed back in the incubator. At the conclusion of the exposure period, 50 μl of media was carefully removed from each well and transferred to a new 96 well clear bottom assay plate. The well exposed to 10× lysis buffer was excluded from the mitochondrial functional assessment. Next, 50 μl of the LDH reaction mixture was added to each well, and the plate was incubated for 30 min at room temperature, protected from light. The reaction was stopped by adding 50 μl of LDH stop solution to each sample. The plate was read using the BioTek Synergy H1 Hybrid reader (BioTek, Winooski, VT, USA) at absorbance of 490 nm and 680 nm. Cell death was calculated as a percentage of total death, relative to maximal cell death induced by treatment with the 10× lysis buffer.