A simple method for isolating rat brain mitochondria with high metabolic activity: effects of EDTA and EGTA.

Javier Iglesias-González, Sofía Sánchez-Iglesias, Andrés Beiras-Iglesias, Ramón Soto-Otero, Estefanía Méndez-Álvarez

Laboratory of Neurochemistry, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain.

Journal of neuroscience methods 2013 Feb 15

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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. Copyright © 2012 Elsevier B.V. All rights reserved.

Citation

Javier Iglesias-González, Sofía Sánchez-Iglesias, Andrés Beiras-Iglesias, Ramón Soto-Otero, Estefanía Méndez-Álvarez. A simple method for isolating rat brain mitochondria with high metabolic activity: effects of EDTA and EGTA. Journal of neuroscience methods. 2013 Feb 15;213(1):39-42