Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation.
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.
Kristián
Tibor
T
Department of Anesthesiology, School of Medicine, University of Maryland, 685 West Baltimore Street, MSTF 534, Baltimore, MD 21201, USA. tkristian@anesthlab.umm.edu
Hopkins
Irene B
IB
McKenna
Mary C
MC
Fiskum
Gary
G
D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D015153:Blotting, Western; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000502:physiology; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008928:Mitochondria / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000502:physiology*; D009584:Nitrogen / Q000502:physiology*; D010101:Oxygen Consumption / Q000502:physiology*; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects
D009419:Nerve Tissue Proteins; D011963:Receptors, GABA-A; D009584:Nitrogen