Protocol for isolating mice skeletal muscle myoblasts and myotubes via differential antibody validation

Figures

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  Figure 1. The process of myoblast isolation from gastrocnemius muscle

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  Figure 2. The process of myotube differentiation from myoblasts and utilization for serum

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  Figure 3. Validation of myotube differentiation via light microscopy (A–C) Myotubes as characterized by (A) Light microscopy, (B) Transmission electron microscopy, and (C) Transfection with adenovirus containing the green fluorescent protein gene (Ad-GFP). (D) Straining with BA-D5-s, D′ SC-71-s, and D″ D3-s to show myosin and desmin.

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  Figure 4. Examples of experiments that may be performed following myotube differentiation and isolation

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  Figure 5. Oxygen consumption rate (OCR) altered in myoblasts and myotubes upon altered insulin stimulation which shows changes in mitochondrial efficiency (A–C) (A) Seahorse plot for primary myoblasts following 2 h of insulin stimulation (B) 4 h of insulin stimulation and (C) 6 h of insulin stimulation. (D–F) (D) Oxygen consumption rate was measured after several inhibitors to measure respiration in primary myotubes after 2 h, (E) 4 h, and (F) 6 h of insulin stimulation. (A′-F′) Basal OCR, which represents respiration under normal, unstressed conditions. (A″-F″) ATP-linked OCR, which is respiration associated with ATP synthesis during oxidative phosphorylation, which is marked by a reduction in OCR due to oligomycin. (A‴-F‴) Maximum OCR, which is the maximal capacity at which mitochondria may utilize oxygen. (A‴’-F‴’) Non-mitochondrial respiration, which can be attributed to factors such as glycolysis or ROS and not due to mitochondrial respiration. These values were compared to the control (blue) in all of these examples. Data are represented as mean ± SEM. N = 6 per treatment, and ∗ indicates p-value < .05.

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  Figure 6. Comparison of mitochondrial fusion proteins following insulin stimulation in primary myoblasts and myotubes (A) Western blotting for mitochondrial fusion protein OPA-1 following 2 h, 4 h, and 6 h of insulin stimulation. (B) OPA-1 levels normalized to Alpha tubulin following insulin stimulation. (C) This was replicated in primary myotubes, as western blotting for mitochondrial fusion OPA1. (D) OPA-1 levels, normalized to Alpha tubulin, in primary myotubes following insulin treatment. Data are represented as mean ± SEM. N = 6 per treatment, and ∗ indicates p-value < .05.

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  Figure 7. TEM quantification of myoblasts (A) Representative transmission electron micrographs from control and (A′) insulin-treated cells, with red arrows showing fused mitochondria. (B–D) (B) Quantifications of number of mitochondria, (C) circularity of mitochondria, (D) and area of mitochondria. (E) Representative transmission electron micrographs from control and (E′) insulin-treated cells, with red arrows showing cristae. (F–H) (F) Quantifications from cristae score, (G) cristae quantity, and (H) cristae area comparing non-insulin and insulin-treated myoblasts. Data are represented as mean ± SEM. Scale bars are 2 μm. Dots show the number of samples. ∗∗ and ∗∗∗∗ indicates p < 0.01 and p < 0.0001, respectively.

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  Figure 8. TEM quantification of myotubes (A) Representative transmission electron micrographs from control and (A′) insulin-treated cells, with red arrows showing fused mitochondria. (B–D) (B) Quantifications of number of mitochondria, (C) circularity of mitochondria, (D) and area of mitochondria. (E) Representative transmission electron micrographs from control and (E′) insulin-treated cells, with red arrows showing cristae. (F–H) (F) Quantifications from cristae score, (G) cristae quantity, and (H) cristae area comparing non-insulin and insulin-treated myotubes. Data are represented as mean ± SEM. Scale bars are 2 μm. Dots show the number of samples. ∗∗ and ∗∗∗∗ indicates p < 0.01 and p < 0.0001, respectively.