title: A simple method for isolating rat brain mitochondria with high metabolic activity: effects of EDTA and EGTA.
abstract: Isolated mitochondria are widely used in metabolic and oxidative stress studies for neurodegenerative diseases. In the present work, the influence of EGTA and EDTA has been tested on a sucrose-based differential centrifugation protocol in order to establish the optimal concentrations to be used in this process. Our results showed alterations in both active and resting respiration, which were dependent on both the addition of EDTA or EGTA to the isolation buffer and the chelator concentration used. However, the addition of chelator to the isolation medium does not modify the mitochondria structure as assessed by both distribution of biological markers and electron micrography in the final pellet. Our results endorse this protocol as the method of choice for metabolic and oxidative stress experiments with fresh isolated rat brain mitochondria.
authors.affiliation: Laboratory of Neurochemistry, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Iglesias-González
authors.forename: Sofía
authors.initials: S
authors.lastname: Sánchez-Iglesias
authors.forename: Andrés
authors.initials: A
authors.lastname: Beiras-Iglesias
authors.forename: Ramón
authors.initials: R
authors.lastname: Soto-Otero
authors.forename: Estefanía
authors.initials: E
authors.lastname: Méndez-Álvarez
mesh_terms: D000818:Animals; D015415:Biomarkers; D001921:Brain / Q000187:drug effects / Q000648:ultrastructure*; D001923:Brain Chemistry / Q000187:drug effects; D002458:Cell Fractionation / Q000379:methods*; D002614:Chelating Agents / Q000494:pharmacology*; D002950:Citrate (si)-Synthase / Q000032:analysis; D004492:Edetic Acid / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology*; D003576:Electron Transport Complex IV / Q000032:analysis; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008297:Male; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010101:Oxygen Consumption / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D015415:Biomarkers; D002614:Chelating Agents; D004533:Egtazic Acid; D004492:Edetic Acid; D007770:L-Lactate Dehydrogenase; D003576:Electron Transport Complex IV; D002950:Citrate (si)-Synthase