Quantifying Myogenic Differentiation

After myoblasts were treated with differentiation medium (DM) containing HG-DMEM supplemented with 2% horse serum (HS, Sigma, USA) for the indicated time, the differentiated myoblasts were stained for MyoG or MEF2C using the primary polyclonal antibody MyoG (sc-12732, 1:150, Santa Cruz) or MEF2C (5030S, 1:400, CST) and the appropriate TRITC-labeled secondary antibody (Jackson Lab, 1:500, USA). The nuclei were stained with DAPI. C2C12 myoblasts with only 1–2 nuclei within a cellular structure were evaluated with MyoG or MEF2C staining. MyoG + or MEF2C + cells were defined as differentiated cells that did not fuse to form myotubes. Myoblasts with 3 or more nuclei in the structure of a cell were defined as myotubes. The number of double-positive nuclei in a high-power field (HPF, 50 μm) was analyzed after double staining with MyoG/DAPI or MEF2C/DAPI. Two individuals who were blinded to the results evaluated the images using ImageJ (Java) software (National Institutes of Health, USA).

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Zhang R.N., Bao X., Liu Y., Wang Y., Li X.Y., Tan G., Mbadhi M.N., Xu W., Yang Q., Yao L.Y., Chen L., Zhao X.Y., Hu C.Q., Zhang J.X., Zheng H.T., Wu Y., Li S., Chen S.J., Chen S.Y., Lv J., Shi L.L, & Tang J.M. (2023). The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy. Journal of Translational Medicine, 21, 173.

Publication 2023

MEF2C MyoG (sc-12732, TRITC-labeled secondary antibody

Antibody Cell nuclei structure Cells Cellular structure Dapi Horse Myoblasts Myotubes Nuclei Serum Tritc

Corresponding Organization :

Other organizations : Hubei University of Medicine, Taihe Hospital, University of Missouri

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Variable analysis

independent variables

Duration of treatment with differentiation medium (DM)

dependent variables

Number of MyoG+ or MEF2C+ cells (differentiated cells that did not fuse to form myotubes)
Number of myotubes (cells with 3 or more nuclei)

control variables

C2C12 myoblasts with only 1–2 nuclei within a cellular structure
HG-DMEM supplemented with 2% horse serum (HS) in the differentiation medium (DM)
Primary antibodies: MyoG (sc-12732, 1:150, Santa Cruz) and MEF2C (5030S, 1:400, CST)
TRITC-labeled secondary antibody (Jackson Lab, 1:500, USA)
DAPI staining for nuclei
Analysis performed by two individuals who were blinded to the results using ImageJ (Java) software

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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