Caspase Assay

Caspase-3 and -7 activity was measured using Caspase-Glo 3/7 (Promega). K562, Jurkat, MCF7 and MDA-MB-231 cells were treated with either 2.5 μM staurosporine, or 100 μg/ml of HEK293 EVs or NK3.3 EVs suspended in PBS at 20% total volume in Eppendorf tubes. 5,000 treated cells in 10 μl were added in triplicate to wells of 384-well plates for each condition and time point. At the appropriate time interval, 10 μl Caspase-Glo 3/7 substrate was added to each well. The plate was incubated for 1 h. For long term detection of caspase activity, 2.5 μM staurosporine or 100 μg/ml of HEK293 EV- or NK3.3 EV-treated K562 cells were seeded in 96-well plates at 50,000 cells/well triplicate for each condition and time point. Caspase activity, quantitated using Caspase-Glo 3/7, was determined using the kit protocol adapted for 5000 cells in 30 μl total volume for each reaction in 384-well plates. Luminescence was measured using the Gen5 microplate reader.