1 9 dimethylmethylene blue chloride

The hydrogel-cell constructs were stained in a solution of Alexa Fluor 488-labeled Phalloidin (0.8 U/ml; Molecular Probes) and 4,6-diamidino-2-phenylindole (DAPI; 2.5 µg/ml; Thermo Fisher) to look at actin filaments and nuclei, respectively. The samples were imaged with an Olympus FluoView confocal microscope with 10X objective with a step size of 10 µm and a total depth of 500 µm. ImageJ software was used to analyze and quantify cell spreading and circularity of the encapsulated cells.
Picogreen/QuanIT DNA Quantification: The DNA quantity of 3 hydrogels per condition and per time point was calculated using the Picogreen DNA QuanIT kit (Invitrogen). The hydrogels were first degraded in a Proteinase-K (0.5 mg/ml; Sigma Aldrich) digestion buffer, mixed with the reagents provided in the kit, and subsequently read using the Qubit Fluorometer.
Dimethylmethylene blue (DMMB) GAG Assay: At each time point, the same digested samples used in the DNA assay were used for the DMMB assay. 1,9-dimethylmethylene blue chloride (Sigma) was dissolved in ethanol and then added to a 0.04 M NaCl/glycine solution, pH 3, for a final concentration of 46 µM DMMB. The samples were loaded into a 96 well plate, along with the DMMB dye solution, and the absorbance was read at 570 nm. The GAG concentration was calculated from a standard curve generated using a serial dilution of chondroitin sulfate (Sigma).