Improving quantification of myotube width and nuclear/cytoplasmic ratio in myogenesis research

Background and Objective: The culture of skeletal muscle cells is particularly relevant to basic biomedical research and translational medicine. The incubation of dissociated cells under controlled conditions has helped to dissect several molecular mechanisms associated with muscle cell differentiation, in addition to contributing for the evaluation of drug effects and prospective cell therapies for patients with degenerative muscle pathologies. The formation of mature multinucleated myotubes is a stepwise process involving well defined events of cell proliferation, commitment, migration, and fusion easily identified through optical microscopy methods including immunofluorescence and live cell imaging. The characterization of each step is usually based on muscle cell morphology and nuclei number, as well as the presence and intracellular location of specific cell markers. However, manual quantification of these parameters in large datasets of images is work-intensive and prone to researcher's subjectivity, mostly because of the extremely elongated cell shape of large myotubes and because myotubes are multinucleated. Methods: Here we provide two semi-automated ImageJ macros aimed to measure the width of myotubes and the nuclear/cytoplasmic localization of molecules in fluorescence images. The width measuring macro automatically determines the best angle, perpendicular to most cells, to draw a profile plot and identify and measure individual myotubes. The nuclear/cytoplasmic ratio macro compares the intensity values along lines, drawn by the user, over cytoplasm and nucleus. Results: We show that the macro measurements are more consistent than manual measurements by comparing with our own results and with the literature. Conclusions: By relying on semi-automated muscle specific ImageJ macros, we seek to improve measurement accuracy and to alleviate the laborious routine of counting and measuring muscle cell features.(c) 2023 Elsevier B.V. All rights reserved.

Automated summary

In this study, two semi-automated ImageJ macros are introduced for measuring the width of myotubes and the nuclear/cytoplasmic localization of molecules in fluorescence images. The macros provide more consistent measurements compared to manual measurements. By using these macros, measurement accuracy can be improved and the laborious task of counting and measuring muscle cell features can be alleviated.