![]() It is important that quantifications are accurate and represent what is biologically observed. 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. ![]() Muscle cell differentiation can be estimated by measuring the fusion index, which is the percentage of nuclei within differentiated myotubes. Skeletal muscle cells go through differentiation where mononucleated myoblasts fuse with each other to form elongated, multinucleated myotubes. For example, a low viability rate after protein depletion may indicate its involvement in cell growth (or apoptosis). Characteristics like cell viability and fusion can be analyzed to determine the role of specific proteins, culturing conditions, or stimuli on the myogenic process. Skeletal muscle cells are easy to culture and thus are often utilized to study different molecular and cellular aspects of myogenesis. Therefore, we propose ViaFuse as a robust and meticulous method to quantify skeletal muscle cell viability and differentiation. Furthermore, ViaFuse is compatible with many computer systems, has a very intuitive interface, and does not require prior complex mathematical knowledge. ![]() Additionally, the ViaFuse macros work with Fiji, an existing imaging software widely used by skeletal muscle researchers. 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. ViaFuse can detect the borders of myotubes and identify nuclear clumps which have been limitations of previous muscle-centric imaging software. We observed a high degree of correlation between all methods of quantification. We 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. 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. Here, we developed a new Fiji macro package called ViaFuse (that stands for viability and fusion) to measure skeletal muscle cell viability and differentiation. Measuring biological features of skeletal muscle cells is difficult because of their unique morphology and multinucleate nature upon differentiation.
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