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bioRxiv · 10.1101/2023.07.11.548594

Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation

Anunciado-Koza, R. V. P.·Guntur, A.·Vary, C. P.·Gartner, C. A.·Nowak, M.·Koza, R. A.
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Abstract

ObjectiveOur goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry. ResultsMitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3-4 hours. Percoll purification from 100-200 mg fresh tissue yielded [~]200-400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9-7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.

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Anunciado-Koza, R. V. P., Guntur, A., Vary, C. P., Gartner, C. A., Nowak, M., Koza, R. A.. 2023-07-11. Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation. https://doi.org/10.1101/2023.07.11.548594

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