Live/Dead staining

At time points of interest, hydrogels were washed 2X with warmed PBS for 5 minutes, incubating at 37°C and 5% CO₂. During incubation, calcein AM and ethidium homodimer-1 from a LIVE/DEAD viability/cytotoxicity kit (Thermo Fisher) were diluted in PBS, 0.5 μL/mL and 2 μL/mL, respectively. The reagents were incubated with the hydrogels for 20–30 minutes at 37°C, and then the hydrogels were washed 2X with warmed PBS for 5 minutes before imaging on an LSM 800 Confocal Microscope (Zeiss). Three hydrogels were imaged per condition and three images were taken per hydrogel with >100 cells counted per condition, and error was calculated from n = 3 hydrogels.