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

BMC Research Notes

Research Note

Carlos A. Gartner¹  Rea Victoria P. Anunciado-Koza¹  Madeleine Nowak²  Anyonya R. Guntur³  Calvin P. Vary³  Robert A. Koza⁴ 

[1] MaineHealth Institute for Research, 81 Research Drive, Scarborough, ME, United States of America;MaineHealth Institute for Research, 81 Research Drive, Scarborough, ME, United States of America;Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME, United States of America;MaineHealth Institute for Research, 81 Research Drive, Scarborough, ME, United States of America;Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME, United States of America;Department of Medicine, Tufts University School of Medicine, Boston, MA, United States of America;MaineHealth Institute for Research, 81 Research Drive, Scarborough, ME, United States of America;Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME, United States of America;Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA, United States of America;

关键词: Percoll purification;    Density gradient;    Mitochondria;    Bioenergetics;    Skeletal muscle;   

DOI  :  10.1186/s13104-023-06519-4

 received in 2022-10-13, accepted in 2023-09-20,  发布年份 2023

来源: Springer

【 摘 要 】

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 h. Percoll purification from 100 to 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 to 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.

【 授权许可】

CC BY   
© BioMed Central Ltd., part of Springer Nature 2023

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