Purification of murine cortex mitochondria using a modified magnetic bead isolation method

We describe a method for purifying murine cortex mitochondria, based on a modification of available technology. The mitochondria isolated and purified using this modified method are viable, without plasma membrane contamination, and suitable for downstream analyses. The method is based on magnetic-activated cell sorting (MACS) technology: extracting mitochondria using a commercially available kit, followed by isolation of mitochondria using antibody-labeled magnetic beads and additional washing steps. We identify the steps crucial for purification from plasma membrane contamination, including optimal starting tissue weight, essential reagents and their preparation, as well as other experimental conditions. The viability of purified mitochondria is assessed using fluorescent imaging. Furthermore, we compare the purity using two additional suboptimal methods in regards to plasma membrane contamination: one that employs an additional commercial kit and protocol, and another that is based on differential centrifugation. The method of choice results in a lower mitochondrial yield, but maximal purity in respect to copurification with other cellular membranes, which is especially important for downstream analyses of high sensitivity. • Successfully overcoming the notoriously challenging step of purification mitochondria free from plasma membrane contamination. • Purity is also achieved in respect to other cellular organelle contamination. • Viable mitochondria is obtained, suitable for downstream analyses.