Mitochondrial Morphology Analysis in H9C2 Cells

To assess mitochondrial morphology by live-cell imaging, H9C2 cardiomyoblasts were plated on coverslips as described above, rinsed with PBS and incubated in phenol-red-free DMEM with 200 nM tetramethylrhodamine methyl ester (TMRM, T5428 Sigma), which accumulates in polarized mitochondria, for a minimum of 20 min. Doxycycline was removed from the medium due to its interfering effects on fluorescence signals [52 (link)]. After wash-out, coverslips were placed inverted on slides. All images were obtained with a Leica TCS SP8 SMD, mounted on a Leica DMI6000 inverted microscope enclosed in an incubator at 37 °C. Images were processed using Leica LAS-X software, and ImageJ, using the FeatureJ plugin (ImageJ 1.45s; National Institutes of Health, Bethesda, USA). Particles were analyzed for area, aspect ratio and circularity as described before [10 (link)].