4.7. Fluorescent recovery after photobleaching (FRAP) experiment

Hydrogels were made using the standard heating–cooling procedure described above. A volume of 200 μL of all formulations were transferred in 35 mm diameter 4 compartment cell culture disk (VWR 391-0225, Greiner bio-one), 1 mL of FITC-labelled dextran solution (3–5 or 70 kDa at 0.1 mg mL⁻¹, Sigma-Aldrich, FD4-250MG and 46945-100MG-F) was added and incubated overnight at 4 °C in the dark. Fluorescent recovery after photobleaching (FRAP) imaging was performed on a Leica TCS SP8 STED using the FRAP modules of Leica Application Suite X software (LAS X FRAP). FRAP bleaching was performed on a z-height of 20 μm in each hydrogel. Parameters were set as followed: bleaching point of 60 μm diameter, bleaching laser at 100%, pre/post bleaching laser at 5%/488 nm/800 gain, and a time per frame of 0.223 s. After 5 frames pre-bleach (1.2 s), samples were bleached for 90 frames (21.2 s) and fluorescent recovery was gathered for 400 frames post-bleaching (110.5 s). 5 different areas within the hydrogels were bleached (N = 5). ROI data were extracted in Fiji ImageJ-win64. Area and mean gray values were obtained for the bleach, total and background ROIs. The obtained values were imported (.cvs files) in the open-source application FrapBot⁶⁸ to obtain the τ_1/2 (half-time) of the fluorescent recovery curve. Obtained half times (τ_1/2) were used to calculate the diffusion coefficients by the Soumpasis equation, with D = diffusion coefficient, r = radius of the bleaching area and τ_1/2 = the halftime of recovery (eqn (4)). Statistical analysis were performed in GraphPad Prism 8.2.0, one-way ANOVA.