Abstract
BackgroundMeasuring biological features of skeletal muscle cells is difficult because of their unique morphology and multinucleate nature upon differentiation. Here, we developed a new Fiji macro package called ViaFuse (that stands for viability and fusion) to measure skeletal muscle cell viability and differentiation. To test ViaFuse, we utilized immunofluorescence images of differentiated myotubes where the capping actin protein of muscle z-line subunit beta (CAPZB) was depleted in comparison with control cells.ResultsWe compared the values achieved using the ViaFuse macros first with manual quantification performed by researchers and second with those obtained utilizing the MATLAB muscle-centric software MyoCount. We observed a high degree of correlation between all methods of quantification.ConclusionsViaFuse can detect the borders of myotubes and identify nuclear clumps which have been limitations of previous muscle-centric imaging software. The ViaFuse macros require little computer power or space to run and user inputs to the ViaFuse macros are minimal, thereby automating the analysis process in a quick, easy, and accurate fashion. Additionally, the ViaFuse macros work with Fiji, an existing imaging software widely used by skeletal muscle researchers. Furthermore, ViaFuse is compatible with many computer systems, has a very intuitive interface, and does not require prior complex mathematical knowledge. Therefore, we propose ViaFuse as a robust and meticulous method to quantify skeletal muscle cell viability and differentiation.
Highlights
Measuring biological features of skeletal muscle cells is difficult because of their unique morphology and multinucleate nature upon differentiation
CAPZB caps the barbed end of actin filaments, preventing the addition or degradation of actin which is critical for ensuring constancy of actin filament length [12]
Cells were stained with DAPI and with an antibody recognizing the myosin heavy chain protein (MYH), which is a marker of muscle cell differentiation (Fig. 1C)
Summary
Measuring biological features of skeletal muscle cells is difficult because of their unique morphology and multinucleate nature upon differentiation. We developed a new Fiji macro package called ViaFuse (that stands for viability and fusion) to measure skeletal muscle cell viability and differentiation. To test ViaFuse, we utilized immunofluorescence images of differentiated myotubes where the capping actin protein of muscle z-line subunit beta (CAPZB) was depleted in comparison with control cells. Skeletal muscle cells are easy to culture and are often utilized to study different molecular and cellular aspects of myogenesis [1]. It is frequently difficult to manually quantify the characteristics of skeletal muscle cells in a dependable and error-free manner because their morphology can vary widely depending on differentiation stage. Some labs utilize various stains to determine myotube density, but these methods are limited to experiments where all myotubes exhibit similar morphology [3, Hinkle et al Skeletal Muscle (2021) 11:28
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