Cellevent caspase3 7 green substrate

A plate-based fluorometric assay (FLUOstar, Omega BMG Labtech) was used to quantify cell permeabilization (5 μM SYTOX Green) and caspase-3 activation (10 μM Asp-Glu-Val-Asp-7-amino-4-methylcoumarin [DEVD-amc]). Briefly, stimulated cells were incubated in the presence of SYTOX Green and DEVD-amc in a CO₂- and temperature-controlled environment that allowed measurement of fluorescent signals over a time span of 3 hr. The maximum slope of fluorescence over time (ΔF/minute) was used as a measure for cell death. Alternatively, the IncuCyte Zoom system (Essenbio) was used to acquire and analyze cell death rates over a period of time. Caspase-3/7 activation was detected using CellEvent Caspase3/7 Green substrate (Invitrogen) according to the manufacturer’s instructions.

Sytox green permeability was used to quantify pyroptosis over time using CellEvent Caspase3/7 green substrate (Invitrogen) according to the manufacturer’s instructions. Data were acquired and analyzed using the IncuCyte Zoom system (Essenbio) over a time span of 25 h. Each condition was run in (technical) duplicates.

Caspase-3/7 activation was detected using CellEvent Caspase3/7 Green substrate (Invitrogen). Data were acquired and analyzed using the Incucyte Zoom system (Essenbio).