Cell Proliferation Assay for Fibroblasts

Cell proliferation assay was performed on NIH-3T3 fibroblast cells (obtained from ATCC (Manassas, VA)) using the method reported by Julie et al^{30 (link)}. The experiment was conducted in triplicate (n = 3). Cells were maintained at 37 °C with 5% CO in complete media consisting of Dulbecco's Modified Essential Medium (DMEM) supplemented with 10% FBS, L-glutamine, and 1% penicillin. At 80–90% confluency, cells were transferred to incomplete media (complete media without 10% FBS). The mitogenic activity of wtFGF1 and the variants was estimated by incubating the cells at varying concentrations (0, 0.4, 2, 10, and 50 ng/mL) of hFGF1. After 24 h of incubation, the number of 3T3 cells were quantified using CellTiter-Glo (Promega, Madison, WI) cell proliferation assay^{20 (link)}.