School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Muscle Nerve. 2011 Sep;44(3):366-70. doi: 10.1002/mus.22056.
C2C12 myoblasts undergo in vitro myogenesis to form protein-rich multinucleated myotubes. Determining the fraction of total nuclei incorporated into myotubes is a commonly used method to quantify the extent of differentiation, but it is labor-intensive and susceptible to operator bias.
We have developed a simple method to quantify myotube formation using micrographs of Jenner-Giemsa-stained C2C12 cultures. Because myotubes are darkly stained by Jenner-Giemsa dyes, the extent of myotube formation correlates with an increase in pixels attributed to the darkest tones. Thus, image histograms were obtained from photographs using ImageJ software, and the sum of the darkest tones was used as a measure of myotube density.
Measurements of myotube density mirrored those of fusion index during C2C12 differentiation and after treatment with prostaglandin D(2) , an inhibitor of C2C12 myogenesis.
We propose this inexpensive, quick, and unbiased method to quantify C2C12 differentiation as a complement of the fusion index analysis.
C2C12 成肌细胞在体外经历肌发生，形成富含蛋白质的多核肌管。测定总核纳入肌管的分数是量化分化程度的常用方法，但它劳动强度大，容易受到操作者的偏见影响。
我们开发了一种使用 Jenner-Giemsa 染色的 C2C12 培养物的显微照片来定量肌管形成的简单方法。由于 Jenner-Giemsa 染料使肌管染色深，因此肌管形成的程度与归因于最暗色调的像素增加相关。因此，使用 ImageJ 软件从照片中获得图像直方图，并将最深色调的总和用作肌管密度的度量。
在 C2C12 分化期间以及在用前列腺素 D2（一种 C2C12 肌发生抑制剂）处理后，肌管密度的测量与融合指数的测量相吻合。
我们提出这种廉价、快速且无偏倚的方法来定量 C2C12 分化，作为融合指数分析的补充。
