Divakaruni 2014 Curr Protoc Toxicol
|Divakaruni AS, Rogers GW, Murphy AN (2014) Measuring mitochondrial function in permeabilized cells using the Seahorse XF Analyzer or a clark-type oxygen electrode. Curr Protoc Toxicol 60:25.2.1-25.2.16.|
Abstract: Measurements of mitochondrial respiration in intact cells can help define metabolism and its dysregulation in fields such as cancer, metabolic disease, immunology, and neurodegeneration. Although cells can be offered various substrates in the assay medium, many cell types can oxidize stored pools of energy substrates. A general bioenergetic profile can therefore be obtained using intact cells, but the inability to control substrate provision to the mitochondria can restrict an in-depth, mechanistic understanding. Mitochondria can be isolated from intact cells, but the yield and quality of the end product is often poor and prone to subselection during isolation. Plasma membrane permeabilization of cells provides a solution to this challenge, allowing experimental control of the medium surrounding the mitochondria. This unit describes techniques to measure respiration in permeabilized adherent cells using a Seahorse XF Analyzer or permeabilized suspended cells in a Hansatech Oxygraph.
• Keywords: Mitochondria, Bioenergetics, Perfringolysin O, Digitonin, XF PMP, C2C12 myoblasts
Labels: MiParea: Respiration, Instruments;methods
Organism: Mouse Tissue;cell: Skeletal muscle, Other cell lines Preparation: Permeabilized cells
Coupling state: LEAK, OXPHOS, ET Pathway: F, N, S, Gp, CIV, ROX