Characterization of myogenesis in C2C12 myoblasts using Flow Cytometry

Abstract

Impaired myogenesis is linked to the muscle wasting and weakness characteristic of sarcopenia and muscular dystrophies. In the past, fusion index, a key biomarker of the myogenic differentiation process, has provided valuable data, but has required labor intensive techniques that often lack precision. Flow Cytometry (FC) provides an accurate and efficient way to characterize cell size, complexity and DNA content with forward scatter (FSC), side scatter (SSC), and propidium iodide staining (FL2), respectively. We propose that as myogenesis progresses, with the accompanying increase in fusion events, FC can be used to detect increasing mitochondrial biogenesis, DNA content and myosin heavy chain in C2C12 mouse skeletal myoblasts. For a more precise comparison of these parameters, we found it useful to synchronize cell cycle at G0/1 through serum deprivation for 24 hours. While still a developing methodology, our preliminary studies, demonstrate that FC could be a valuable method to quantify variations in cell size, character and DNA content in myoblasts and myotubes. This method promises to provide a more reliable characterization of myogenic differentiation to advance this field of research. Support: NIH‐National Institutes of Aging P01 AG039355 and Missouri Life Sciences Research Board (MB).