Quantification of C2C12 Myoblast Differentiation by Flow Cytometry

C2C12 myoblasts are widely used as an in vitro model to study skeletal muscle cell differentiation. Here, we update our previously published flow cytometry-based protocol to quantify C2C12 cell differentiation by measuring myosin heavy-chain (MyHC) marker expression. We explain the procedures for daily maintenance, transfection, induction of C2C12 cell differentiation, immunolabeling, and flow cytometry acquisition settings. We emphasize the importance of optimized cell seeding density to enhance transfection or differentiation efficiency and clarify the critical role of the correct cell population gate to accurately quantify MyHC-positive cells. Both the effect of drug treatments and genetic manipulations can be analyzed in this way. As an example, we quantify the disruption and rescue of differentiation only of the transformed, GFP-positive cell population after expression of mEGFP-tagged oncogenic K-Ras.