Isolation of Intact Mitochondria From <em>Drosophila melanogaster</em> and Assessment of Mitochondrial Respiratory Capacity Using Seahorse Analyzer

Analysis of mitochondrial function has broad applicability in many research specialties. Neurodegenerative disorders such as chemotherapy-induced peripheral neuropathy (CIPN) often exhibit damaged mitochondria or reduced mitochondrial respiratory capacity. Isolation of intact mitochondria for protein analysis or respiration measurements has been previously reported in numerous model organisms. Here, we describe an adaptation of previous protocols to isolate intact functional mitochondria from <i>Drosophila melanogaster</i> for use in a model of CIPN. Whole <i>Drosophila</i> are ground in isolation buffer, and mitochondria are purified using differential centrifugation through a sucrose and mannitol solution. The intact mitochondria are plated as a monolayer for measurements of mitochondrial oxygen consumption rates and response to inhibitor compounds on an Agilent Seahorse analyzer. This experimental protocol is quick and yields a purified population of intact mitochondria that may be used for functional assays for several hours after isolation. The isolated mitochondria may be used for respiration measurements, which reflect their health, and stored for protein or genetic analysis. Mitochondrial populations from multiple strains or treatment groups can be easily compared simultaneously. The rapid biochemical assessment of mitochondria, in combination with the utility of <i>Drosophila</i> as an in vivo genetic model system, offers great potential for researchers to probe the impact of genetics and pharmacologic interventions on mitochondrial respiratory capacity. Key features • This protocol describes rapid isolation of intact, functional mitochondria that may be used for respiration measurements or other biochemical analyses. • Mitochondria isolated from <i>Drosophila</i> are assessed in an Agilent Seahorse analyzer utilizing multiple substrates and electron transport chain inhibitors to fully characterize mitochondrial respiratory capacity. • This protocol is optimized to use <i>Drosophila</i> for easy in vivo genetic and pharmacologic manipulation, and assessment of the impact on mitochondrial function.