Fluorescent characterization of differentiated myotubes using flow cytometry.

Area of Science:

• Cell Biology
• Skeletal Muscle Research
• Flow Cytometry Applications

Background:

• Flow cytometry is standard for assessing myoblast populations.
• A comprehensive gating strategy for differentiated myotubes using flow cytometry is lacking.
• Myotubes exhibit significant metabolic and protein changes compared to myoblasts.

Purpose of the Study:

• To establish the utility of flow cytometry for analyzing both myoblasts and myotubes.
• To characterize morphological and metabolic differences between myoblasts and myotubes.
• To develop a gating strategy for myotube identification within heterogeneous cell suspensions.

Main Methods:

• C2C12 murine cells were analyzed for morphology and metabolic reprogramming using flow cytometry.
• Laser scatter (FSC/SSC) measured cell morphology; fluorescent probes quantified mitochondrial mass, ROS, and DNA content.
• Immunophenotyping for myosin heavy chain (MyHC) confirmed differentiation; viability was assessed using Annexin V/propidium iodide.

Main Results:

• Myotubes showed significantly higher mitochondrial mass (159%), ROS production (303%), DNA content (18%), and MyHC expression (147%) compared to myoblasts.
• Laser scatter indicated similar sizes for myoblasts and myotubes, but 'rolled-up' myotube morphology altered scatter properties.
• Myotubes displayed greater heterogeneity, with two distinct sub-populations identified, one viable and one likely containing apoptotic bodies.

Conclusions:

• Flow cytometry is effective for analyzing myotubes, with increased mitochondrial mass, ROS, and DNA content being key identifiers correlating with MyHC expression.
• Myotube 'rolling up' during analysis complicates size determination via laser scatter, necessitating alternative identification strategies.
• Acoustic focusing may be superior to hydrodynamic focusing for resolving distinct myotube sub-populations due to their heterogeneity.