{"doi":"10.1101/2023.07.11.548594","title":"Purification of functional mouse skeletal muscle mitochondria using Percoll density gradient centrifugation","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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":400079,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.942,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":266143,"name":"Anyonya R. Guntur","orcid":"0000-0001-6366-200X","position":1,"is_corresponding":false},{"id":323271,"name":"Calvin Vary","orcid":"0000-0001-8793-0128","position":2,"is_corresponding":false},{"id":992978,"name":"Carlos A. Gartner","orcid":null,"position":3,"is_corresponding":false},{"id":1114020,"name":"Madeleine Nowak","orcid":null,"position":4,"is_corresponding":false},{"id":376875,"name":"Robert A. Koza","orcid":"0000-0001-6994-7268","position":5,"is_corresponding":false},{"id":1114019,"name":"Rea Victoria P. Anunciado-Koza","orcid":null,"position":0,"is_corresponding":true}],"reference_count":14,"raw_metadata":null,"created_at":"2026-07-19T01:20:00.023537Z","pmid":"37503222","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}