Purification of functional mouse skeletal muscle mitochondria using percoll density gradient centrifugation.
Bioenergetics
Density gradient
Mitochondria
Percoll purification
Skeletal muscle
Journal
BMC research notes
ISSN: 1756-0500
Titre abrégé: BMC Res Notes
Pays: England
ID NLM: 101462768
Informations de publication
Date de publication:
30 Sep 2023
30 Sep 2023
Historique:
received:
13
10
2022
accepted:
20
09
2023
medline:
2
10
2023
pubmed:
1
10
2023
entrez:
30
9
2023
Statut:
epublish
Résumé
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. 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.
Identifiants
pubmed: 37777771
doi: 10.1186/s13104-023-06519-4
pii: 10.1186/s13104-023-06519-4
pmc: PMC10544150
doi:
Substances chimiques
Percoll
65455-52-9
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
243Subventions
Organisme : NIGMS NIH HHS
ID : P20 GM121301
Pays : United States
Commentaires et corrections
Type : UpdateOf
Informations de copyright
© 2023. BioMed Central Ltd., part of Springer Nature.
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