Live dead kit

For staining of healthy cells, we incubated cells with the calcein-green component (1/2000) of the LIVE/DEAD Kit (Molecular Probes, Darmstadt, Germany) for 10 min at 37 °C followed by immunocytochemistry (or direct imaging), which was performed according to standard procedures.^{37 (link)} Fluorescence imaging was carried out on a Zeiss Axiovert 200 M inverted fluorescence microscope (Zeiss, Oberkochen, Germany) equipped with a 12-bit monochrome CoolSNAP ES2 CCD camera (Photometrics, Tucson, AZ, USA) using the MetaVue and Metamorph (analysis) software (Visitron Systems, Puchheim, Germany). Aβ-induced cell death was studied by using the LIVE/DEAD Kit (Molecular Probes) according to the manufacturer's instructions.

iECs were encapsulated into hydrogels as described above. To assess the proper incubation time for the photoinitiator, the gels were incubated with a photoinitiator for 5, 10, and 30 min. After incubation, gels were washed using endothelial cell growth media and incubated in EC diff media for another 24 h. Cell viability was assessed using calcein‐AM and ethidium homodimer‐1 (Live/Dead kit, Thermo Fisher, L3224).
To determine cell viability for stiffness experiments, iECs were encapsulated into hydrogels, allowing network formation for 48 h. The networks were then incubated with photoinitiator solution for 10 min and crosslinked for the indicated time. After crosslinking, the constructs were extensively washed with high VEGF media containing 0.2% DMSO and cultured in high VEGF media containing 0.2% DMSO for another 24 h. Cell viability was assessed using calcein‐AM and ethidium homodimer‐1 (Live/Dead kit, Thermo Fisher, L3224) and LSM780 confocal microscope (Zeiss) and analyzed using ImageJ software. ^[71]