Iglesias-Gonzalez 2013 J Neurosci Methods

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Iglesias-Gonzalez J, Sanchez-Iglesias S, Beiras-Iglesias A, Soto-Otero R, Mendez-Alvarez E (2013) A simple method for isolating rat brain mitochondria with high metabolic activity: Effects of EDTA and EGTA. J Neurosci Methods 213:39-42.

» PMID: 23261657

Iglesias-Gonzalez J, Sanchez-Iglesias S, Beiras-Iglesias A, Soto-Otero R, Mendez-Alvarez E (2013) J Neurosci Methods

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.

Keywords: Mitochondria, Brain, EDTA, EGTA, Isolation procedure, Mitochondrial respiration, Oxidative stress

O2k-Network Lab: ES Santiago De Compostela Mendez-Alvarez E


Labels:

Stress:Mitochondrial disease  Organism: Rat  Tissue;cell: Nervous system  Preparation: Isolated mitochondria 


Coupling state: LEAK, OXPHOS  Pathway:HRR: Oxygraph-2k 


Comments

by Erich Gnaiger (2013-01-31)

  • Electron transport system activity cannot be quantified in the active state (State 3; OXPHOS or State P), but uncoupler titrations are required to determine the ET-capacity (E), for evaluation of a possible difference between E and P. In many types of mitochondria, the P/E ratio is >1.0 [1,2].
  1. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837-1845.
  2. Gnaiger E (2012) Mitochondrial Pathways and Respiratory Control. An Introduction to OXPHOS Analysis. Mitochondr Physiol Network 17.18. Oroboros MiPNet Publications, Innsbruck: 64 pp.