A Novel Mitophagy Assay for Skeletal Myotubes

Michael Webb, Jyoti Malhotra, Chui-Se Tham, Matthew Goddeeris, Douglas W McMillin and Effie Tozzo
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Effie Tozzo: Mitobridge Inc, USA

Open Access Journal of Neurology & Neurosurgery, 2017, vol. 4, issue 4, 79-86

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.

Keywords: Open Access Journal of Neurology; Journal of Neurology & Neurosurgery; Open Access Journal; juniper publishers; neurology open access journals; journal of neurology; neurology indexed journals; neurology scholarly journals; journal of neurology; neurology journals impact factor; juniper publishers reivew (search for similar items in EconPapers)
Date: 2017
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Persistent link: https://EconPapers.repec.org/RePEc:adp:joajnn:v:4:y:2017:i:4:p:79-86

DOI: 10.19080/OAJNN.2017.04.555649

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