Celldiscoverer 7 imaging system

Induction of apoptosis was performed using a combination of 20 ng/ml recombinant mouse or human TNF (PeproTech), 10 μg/ml CHX (Selleckchem), and 5 μM LCL-161 (MedChemExpress) as reported^{23 (link)}. The concentrations of GSK’872 (Selleckchem) and Z-IETD-FMk (Selleckchem) used were 10 μM as reported^{23 (link)}. To induce necroptosis, the same concentrations were used, with the addition of 20 μM zVAD-fmk (Selleckchem) as reported^{23 (link)}. At the endpoint of the experiment, cells were stained with the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) following the manufacturer’s instructions and analyzed using a BD LSRFortessa flow cytometer (BD Biosciences). To detect the cell-surface level of TNFR1, cells were stained with APC-CD120a (Biolegend). Flow cytometry data were analyzed by Flowjo_V10. For live imaging of organoid cell death, the organoids were incubated with CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific) and PI (BD Biosciences) for 30 minutes at 37 °C. Images were acquired using the ZEISS Celldiscoverer 7 imaging system (ZEISS).

On day 14, cultures of 600 k HPMEC in ECM hydrogel were stained with Hoechst, PI, and Calcein AM as described above, and optical sections were obtained by scans using the Zeiss Cell discoverer 7 imaging system (Zeiss, Jena, Germany). Optical settings were an objective lens magnification of 5× and an Optovar magnification of 1×. The interval of the scanning was 325 µm. Detection wavelengths (excitation–emission) were 400–565 nm for Hoechst, 400–575 nm for Calcein AM, and 575–700 nm for Texas Red. Image stacks and videos were generated using Zeiss Zen 3.3 software.