Flow Cytometric Methods for ROS Quantification in Skeletal Muscle Myotubes

Flow cytometry is a high-throughput technique which can be used to analyze and quantify characteristics of individual cells. Flow cytometry hydrodynamically focuses cells past a series of size and fluorescent detectors to characterise cellular morphology and phenotype. This process of hydrodynamic focusing has previously been shown to be incompatible with myotubes due to their larger size relative to other cell types. Here, we describe the culture and differentiation of myotubes and how myotubes can be analyzed via flow cytometry to quantify changes in cellular and mitochondrial-specific ROS production.