ArticlebioRxiv : the preprint server for biology2023
Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation.
Article in bioRxiv : the preprint server for biology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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6 authors at 3 institutions in 1 country.
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Abstract
Objective: Our 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. Results: Mitochondria 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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