Collagen coating

Collagen coating of plates was performed inside a cell culture hood. Initially, Collagen (Thermo Fisher Scientific, Catalogue #A1048301), sterile 10X phosphate buffered saline (Thermo Fisher Scientific, Catalogue #AM9625), sterile distilled water (dH2O), and sterile 1 N NaOH (Merck, Catalogue # S2770-100ML) was thawed on ice. For our experiment we used final Collagen concentration of 1 mg/ml and 0.5 mg/ml. Total volume of collagen stock required for preparation of each dilution was calculated as per manufacturers protocol (Thermo Fisher Scientific, Catalogue #A1048301)

Subsequently, in a sterile tube, dH2O, 1 N NaOH, and 10X PBS were mixed. The estimated amount of Collagen was slowly pipetted into the tube, with multiple rounds of pipetting the solution up and down to achieve a homogeneously mixed solution. The resulting mixture was checked to ensure that it achieved a pH of 6.5–7.5 (optimal pH is 7.0). The diluted Collagen solution was then dispensed carefully into the plates (500 μl per well of a standard 6 well tissue culture plates), on ice. The ice was used to ensure uniform coating of the plate with the cold temperature thwarting rapid gelling at room temperature. The plates were then incubated at 37 °C in a humidified incubator for 30–40 min until a firm gel was visually observed. Lastly, the wells were rinsed twice with 1X PBS and cell culture medium before seeding of cells.