Isolation of mitochondria from the CNS

Abstract

This unit contains a protocol describing the isolation of brain mitochondria by using discontinuous Percoll gradient centrifugation. The Percoll density gradient centrifugation separates synaptosomes, myelin, and free nonsynaptic mitochondria released from cells during tissue homogenization into individual fractions. Mitochondria entrapped in synaptosomes (synaptic mitochondria) can be liberated using nitrogen cavitation and then further purified by Percoll gradient centrifugation. These methods yield mitochondria that exhibit good respiratory coupling and high respiratory rates.

Figure 1

Flow schema of the separation procedure for mitochondria and synaptosomes from brain tissue. Following homogenization of brain tissue and low speed centrifugation the crude mitochondrial sample is fractionated into non-synaptic mitochondria and synaptosomes by Percoll gradient centrifugation.

Figure 2

Schematic illustration of the Percoll gradient and the distribution of brain homogenate fractions following centrifugation.

Figure 3

Flow schema of the protocol for the isolation of synaptic mitochondria from synaptosomes by using Nitrogen cavitation. The numbers on the upper tube represent Percoll solutions concentrations used to create the gradient.