C2C12 Cell Culture Growth and Differentiation Protocols

C2C12 cells were obtained from the American Type Culture Collection (ATCC) and were initially provided in DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 10% Dimethylsulphoxide (DMSO). The cells were thawed in a 37°C water bath and washed with 1X PBS to remove excess DMSO. Following this, the cells were centrifuged at 750 x g, resuspended in growth media (DMEM supplemented with 10% FBS and 2% Pen-Strep), and plated on 10 cm tissue culture plates. The cells were incubated at 37°C with 5% CO₂. Passaging was performed when cells reached 70% confluency, and the media was replaced every 48 hours to maintain cell growth. For differentiation, cells were induced under low serum conditions. The growth media was aspirated, cells were washed with 1X PBS, and differentiation media (DMEM supplemented with 2% Horse Serum and 2% Pen-Strep) was added. Cells were differentiated according to experimental protocols³.