Abstract
Identification of differentiating muscle cells generally requires fixation, antibodies directed against muscle specific proteins, and lengthy staining processes or, alternatively, transfection of muscle specific reporter genes driving GFP expression. In this study, we examined the possibility of using the robust mitochondrial network seen in maturing muscle cells as a marker of cellular differentiation. The mitochondrial fluorescent tracking dye, MitoTracker, which is a cell-permeable, low toxicity, fluorescent dye, allowed us to distinguish and track living differentiating muscle cells visually by epi-fluorescence microscopy. MitoTracker staining provides a robust and simple detection strategy for living differentiating cells in culture without the need for fixation or biochemical processing.
Highlights
Much of our extensive knowledge of the molecular and cell biology of skeletal muscle differentiation is due to the availability of immortalized cell lines such as the C2 cell line, with its sub-variants including the C2C12 line
In order to test mitochondrial reactivity in differentiating cells to the MitoTracker, C2C12 cells were induced to differentiate in differentiation medium (DM; 2% FBS containing DMEM) for 4 days
We report a simple, quick and effective method to identify differentiating muscle cells based on mitochondrial activity with a cell-permeable fluorescent dye, MitoTracker
Summary
Much of our extensive knowledge of the molecular and cell biology of skeletal muscle differentiation is due to the availability of immortalized cell lines such as the C2 cell line, with its sub-variants including the C2C12 line. C2 cells were originally established from adult satellite cells [1,2]. These cells can proliferate with high mitogenic stimuli and form multi-nucleated myotubes (MTs) readily upon reduction of mitogens. The differentiation process is not fully synchronized, and due to stochastic reasons, a significant portion of the population does not form differentiated MTs, remaining in a quiescent mono-nucleated state [3]. The ability to separate these populations would be a great advantage in characterizing the molecular events during muscle differentiation. Fixation of the cells or transfection methods may limit downstream applications
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