Cells were harvested from alginate hydrogels by dissolving the gels using 50 mM EDTA. Cell cycle progression was then analyzed using flow cytometry (Stanford Shared FACS Facility, Scanford). Briefly, all cells harvested from the alginate gels were washed with DPBS and then fixed with cold 70% (v/v) ethanol overnight at 4°C. Cells were then resuspended in DPBS containing RNaseA (a concentration of 20 μg/ml) and propidium iodide (a concentration of 20 μg/ml) to stain DNA for 20 min. The histogram of DNA content was obtained by gating cell population on a fluorescence intensity–versus–forward scatter dot plot. Once viable cells were gated, the fraction of cells at each cell cycle was quantified by fitting the experimentally obtained histogram with the Watson model in FlowJo version 10.2 software.

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