Khattar 2016 J Neurosci Methods

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Khattar NK, Yablonska S, Baranov SV, Baranova OV, Kretz ES, Larkin TM, Carlisle DL, Richardson RM, Friedlander RM (2016) Isolation of functionally active and highly purified neuronal mitochondria from human cortex. J Neurosci Methods 263:1-6.

» PMID: 26808294

Khattar NK, Yablonska S, Baranov SV, Baranova OV, Kretz ES, Larkin TM, Carlisle DL, Richardson RM, Friedlander RM (2016) J Neurosci Methods

Abstract: Functional and structural properties of mitochondria are highly tissue and cell dependent, but isolation of highly purified human neuronal mitochondria is not currently available.

We developed and validated a procedure to isolate purified neuronal mitochondria from brain tissue. The method combines Percoll gradient centrifugation to obtain synaptosomal fraction with nitrogen cavitation mediated synaptosome disruption and extraction of mitochondria using anti mitochondrial outer membrane protein antibodies conjugated to magnetic beads. The final products of isolation are non-synaptosomal mitochondria, which are a mixture of mitochondria isolated from different brain cells (i.e. neurons, astrocytes, oligodendrocytes, microglia) and synaptic mitochondria, which are of neuronal origin. This method is well suited for preparing functional mitochondria from human cortex tissue that is surgically extracted.

The procedure produces mitochondria with minimal cytoplasmic contaminations that are functionally active based on measurements of mitochondrial respiration as well as mitochondrial protein import. The procedure requires approximately four hours for the isolation of human neuronal mitochondria and can also be used to isolate mitochondria from mouse/rat/monkey brains.

This method will allow researchers to study highly enriched neuronal mitochondria without the confounding effect of cellular and organelle contaminants.

Copyright © 2016 Elsevier B.V. All rights reserved.

Keywords: Differential centrifugation, Functional, Human cortex, Isolation, Mitochondria, Neurons, Non-synaptosomal mitochondria, Percoll, Purified, Synaptosome

O2k-Network Lab: US PA Pittsburgh Baranov SV

Labels: MiParea: Respiration, Instruments;methods 

Organism: Human, Mouse  Tissue;cell: Nervous system  Preparation: Isolated mitochondria  Enzyme: Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase 

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