Quantifying EGF Release from Hydrogels

HP-B hydrogel was pre-mixed with 50 ng ml^–1 EGF. 1.5 mL of this pre-mixed hydrogel was added to a 12-well plate (n = 3) and allowed to gelate for about 4 to 5 hours at room temperature. Following this, 1.5 mL of serum-free, EGF-free growth media was added on top of the hydrogel in each of the wells and incubated at 37 °C. The supernatant was collected, and the wells were replenished with 1.5 ml of fresh serum-free, EGF-free media every 24 hours for a total of 72 hours. The collected supernatants were analyzed for total EGF content, using EGF Human ELISA kit (Invitrogen, KHG0061). Previous studies have validated a normalization factor to account for the degradation of the protein in heparin-based hydrogel, that was used to post-process our data from ELISA.36 (link)