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Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation
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 skele...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10544150/ https://www.ncbi.nlm.nih.gov/pubmed/37777771 http://dx.doi.org/10.1186/s13104-023-06519-4 |
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author | Anunciado-Koza, Rea Victoria P. Guntur, Anyonya R. Vary, Calvin P. Gartner, Carlos A. Nowak, Madeleine Koza, Robert A. |
author_facet | Anunciado-Koza, Rea Victoria P. Guntur, Anyonya R. Vary, Calvin P. Gartner, Carlos A. Nowak, Madeleine Koza, Robert A. |
author_sort | Anunciado-Koza, Rea Victoria P. |
collection | PubMed |
description | 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 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13104-023-06519-4. |
format | Online Article Text |
id | pubmed-10544150 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-105441502023-10-03 Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation Anunciado-Koza, Rea Victoria P. Guntur, Anyonya R. Vary, Calvin P. Gartner, Carlos A. Nowak, Madeleine Koza, Robert A. BMC Res Notes Research Note 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 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13104-023-06519-4. BioMed Central 2023-09-30 /pmc/articles/PMC10544150/ /pubmed/37777771 http://dx.doi.org/10.1186/s13104-023-06519-4 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Note Anunciado-Koza, Rea Victoria P. Guntur, Anyonya R. Vary, Calvin P. Gartner, Carlos A. Nowak, Madeleine Koza, Robert A. Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation |
title | Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation |
title_full | Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation |
title_fullStr | Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation |
title_full_unstemmed | Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation |
title_short | Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation |
title_sort | purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation |
topic | Research Note |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10544150/ https://www.ncbi.nlm.nih.gov/pubmed/37777771 http://dx.doi.org/10.1186/s13104-023-06519-4 |
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