Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation - PubMed (original) (raw)

Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation

Tibor Kristián et al. J Neurosci Methods. 2006.

Abstract

To study neurons or glia-specific mitochondria one needs to isolate these organelles from primary neuronal or astrocytic cell culture. This work provides novel method for isolation of functional and morphologically intact mitochondria from neurons and astrocytes in cell cultures. In the first step, mitochondria are released from cells by disruption of cell membranes using a nitrogen cavitation technique. This technique is based on rapid decompression of a cell suspension from within a pressure vessel. Mitochondria released from cell bodies are then separated from the rest of cell homogenate by Percoll gradient centrifugation. This is a relatively rapid technique that yields to very well coupled mitochondria that exhibited functional and morphological characteristics comparable to mitochondria isolated from brain tissue using common techniques. This technique thus will allow examination of mitochondria that are exclusively cell specific in origin.

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Figures

Fig. 1

Flow scheme of the isolation procedure using the nitrogen cavitation technique. The number in parenthesis represents the percentage of Percoll used to form the gradient for separation of mitochondria from astrocytes.

Fig. 2

Mitochondria isolated from neuronal or astrocytic culture display high respiratory control ratios. Neuronal (A) or astrocytic (B) mitochondria (0.25 mg) were added to a Clark-type oxygen electrode chamber maintained at 37 °C. The respiratory medium containing ADP (0.5 mM) stimulated the mitochondrial respiration (State 3). After about 2 min the ADP-induced oxygen consumption was stopped by addition of 1.25 μg/ml oligomycin, a mitochondrial ATP-synthase inhibitor. Rates of oxygen consumption following oligomycin addition (State 4o) represent the resting state of respiration dependent only on the unspecific leak of hydrogen ions across the inner mitochondrial membrane. The respiratory control ratio (State 3 rate divided by State 4o rate) was about 9–10 for both astrocytic and neuronal samples suggesting functionally well preserved mitochondria.

Fig. 3

Electron micrograph of neuronal (A) and astrocytic (B) mitochondria. The outer and inner mitochondrial membranes are clearly identifiable. The mitochondrial matrix is electron dense with no signs of swelling or damage. Interestingly, the morphology of the cristea in neuronal mitochondria (A) have a parallel alignment, whereas in astrocytic mitochondria the cristea are randomly oriented. Both neuronal and astrocytic mitochondrial samples contain cell debris visible as diffuse dark material and small vesicles. The scale bars represent 1 μm.

Fig. 4

Immunoblots showing the relative distribution of mitochondrial and non-mitochondrial proteins in astrocytic, neuronal, and non-synaptic mitochondria. The immunoreactivity of subunits of OXPHOS complexes was lower in neuronal mitochondria when compared to astrocytic and non-synaptic mitochondria. However, the cytosolic protein β-actin was not detected in non-synaptic mitochondria, while the neuronal and astrocytic mitochondria expressed some contamination by this protein. Immunoreactivity to the peripheral benzodiazepine receptor (PBR) was evident only in the astrocytic sample, completely lacking in neuronal mitochondria, and slightly detectable in the non-synaptic mitochondrial sample.

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Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation - PubMed (original) (raw)