Preparation and respirometric assessment of mitochondria isolated from skeletal muscle tissue obtained by percutaneous needle biopsy.
Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 μg of mitochondria indicate that 1.0 μg is sufficient to measure respiration and that 5.0 μg provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.
Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine.
Manish S
MS
Bharadwaj
Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine.
Daniel J
DJ
Tyrrell
Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine.
Mary F
MF
Lyles
Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine.
Jamehl L
JL
Demons
Seahorse Biosciences.
George W
GW
Rogers
Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest School of Medicine; amolina@wakehealth.edu.
Anthony J A
AJ
Molina
D001707:Biopsy, Needle / Q000379:methods*; D006801:Humans; D008931:Mitochondria, Muscle / Q000737:chemistry* / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000502:physiology*