Inducing Cell Death in FIECs and HT3B3 Cells

Death was induced in FIECs by incubation with 1 µm z-VAD-fmk and 1 µm staurosporine (TOCRIS Bioscience) in DMEM plus 20% FBS at 37 °C in 5% CO₂ for 8 h^{59 (link)}. FIECs were stained with 500 nm PI and 2 µg/ml H33342 for 5 min in the dark prior to visualization by confocal microscopy.
RIP1K-dependent necroptosis was induced in HT3^B3-WT and HT3^B3-KO cells using by pretreating cells with 100 nm BV6, 500 nm qVD-OPh, and 1 µm cyclohexamide at 37 °C in 5% CO₂^{125 (link)}. After 1 h incubation, human TNFα was added to a final concentration of 10 ng/ml and incubated for 12–16 h at 37 °C in 5% CO₂. Cell death was measured by uptake of 100 nm Sytox™ green imaged on the BioTek Cytation^™ 5.