FRAP was performed to measure HELP hydrogel diffusivity as previously described (89 (link)). Briefly, 30 μl of gels was formed at the bottom of a clear-bottom, half-area, black 96-well plate (Greiner Bio-One). Once gels had formed, solutions of fluorescein isothiocyanate–labeled dextrans (4 mg/ml; Sigma-Aldrich) of varying molecular weights (10, 20, 40, 70, 150, and 250 k) were added to each respective well and allowed to incubate at 37°C overnight. Using a confocal microscope (Leica SPE), a 100 μm by 100 μm area of each hydrogel was photobleached using a 488-nm laser at 100% intensity for 1 min. Immediately following photobleaching, fluorescence recovery into the photobleached region was monitored for 4 min (1 frame/s) at 10% laser intensity. Images were analyzed using open source MATLAB code that was previously published (90 (link)).
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