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A Novel Mitophagy Assay for Skeletal Myotubes

Author

Listed:
  • Michael Webb
  • Jyoti Malhotra
  • Chui-Se Tham
  • Matthew Goddeeris
  • Douglas W McMillin
  • Effie Tozzo

    (Mitobridge Inc, USA)

Abstract

We developed a simple ELISA technique as a quantitative in vitro assay for mitophagy in post mitotic skeletal muscle cells. We labeled the mitochondrial DNA of C2C12 myotubes cells by allowing incorporation of the thymidine analogue bromodeoxyuridine (BrdU) and following the decay of the label with time under basal and stimulated conditions. BrdU was incorporated into differentiated C2C12 myotubes at significant levels only after pharmacological treatment was used to increase the rate of mtDNA synthesis. PGC1α transduction, stimulation of AMPK, or inhibition of ERK1/2, all previously reported to increase the rate of mtDNA synthesis, induced BrdU incorporation at 4-10 fold over background during a 2 hour incorporation period. Using AMPK stimulation to stimulate initial DNA labeling we confirmed by both microscopy and cytometry methods that BrdU was incorporated into mitochondria and not into nuclei.

Suggested Citation

  • Michael Webb & Jyoti Malhotra & Chui-Se Tham & Matthew Goddeeris & Douglas W McMillin & Effie Tozzo, 2017. "A Novel Mitophagy Assay for Skeletal Myotubes," Open Access Journal of Neurology & Neurosurgery, Juniper Publishers Inc., vol. 4(4), pages 79-86, July.
    • Handle: RePEc:adp:joajnn:v:4:y:2017:i:4:p:79-86
    DOI: 10.19080/OAJNN.2017.04.555649
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    References listed on IDEAS

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    1. Konstantinos Palikaras & Eirini Lionaki & Nektarios Tavernarakis, 2015. "Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans," Nature, Nature, vol. 521(7553), pages 525-528, May.
    2. Jonathan R. Friedman & Jodi Nunnari, 2014. "Mitochondrial form and function," Nature, Nature, vol. 505(7483), pages 335-343, January.
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