Table 1.

Difficulties in Myotube Size Analysis

Problem	Implication for Myotube Size Analysis	Improvements Afforded by the Present Photoshop Technique
Many myotubes are noncylindrical	Diameter measurements would be grossly inaccurate.	Enables fast, accurate and nonbiased surface area measurements that are not dependent on cell morphology.
Large variability in myotube sizes	Averaging myotube sizes may conceal an experimental finding at a particular point within the size range.	Photoshop technique can be applied to individual myotubes in a FOV or to all myotubes in a FOV simultaneously.
Nuclear clustering	It is difficult or impossible to calculate fusion index, myonuclear incorporation, and myonuclear domains.	Area measurements are less time-consuming and more direct measures of myotube size than fusion index or nuclear incorporation.
Presence of mononuclear cells in myotube cultures	High-throughput analysis based on proteins associated with terminal differentiation (e.g., MHC) may pick up myoblasts, which express these markers.	There is easy removal of debris and interfering/overlapping cells from one or all color channels.
Formation is highly dependent on seeding density	High plating densities improve the statistical likelihood of MPC fusion and myotube formation. This may overwhelm the effect of an experimental agent on fusion, thus concealing its influence. The inverse is true for low seeding densities.	Myotube formation could be normalized to total area of nuclei/FOV, although nuclear clustering may complicate this.
Whole myotubes often extend beyond limits of image field	Many experimenters may be reporting the measurement of myotubes when they are in fact measuring segments of myotubes or “myosegments”.	No immediate solution is provided by the current technique, but it can easily be applied to the measurement of both whole myotubes and myosegments.
Experimenter bias	Techniques such as diameter measurements, manual tracing of myotubes, and selection of “small” or “large” myotubes are all open to bias.	The creation of a specific color range selection mask for MHC allows accurate, fast, and objective measurement of myotube/segment areas.

MHC, myosin heavy chain; MPC, muscle precursor cell; FOV, field of view.