Efficient Isolation of Pure and Functional Mitochondria from Mouse Tissues Using Automated Tissue Disruption and Enrichment with Anti-TOM22 Magnetic Beads

To better understand molecular mechanisms regulating changes in metabolism, as observed e. g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique

Liver, muscle and brain mitochondria isolated using anti-TOM22 magnetic beads show reliable mitochondrial function.

<p>Oxygen consumption of isolated <b>A</b> liver, <b>B</b> muscle and <b>C</b> brain mitochondria were measured by a Clark electrode. Malate+pyruvate (MP) and malate+glutamate (MG) served as specific substrates for complex I, succinate (S, for complex II and glycerol-3-phosphate (GP) for complex III, respectively. ADP was given to malate+pyruvate to determine state III respiration via complex I. Oligomycin and CCCP were used with succinate to assess state IVo and uncoupled respiration, respectively. The given oxygen consumption units are nmol oxygen/min/mg mitochondria. Data are represented as means of three independent experiments ± SD.</p

Liver mitochondria isolated by magnetic beads have higher oxygen consumption rates than mitochondria isolated by differential centrifugation.

<p>Oxygen consumption rates (OCRs) were determined using an XF24 Extracellular Flux Analyzer with the given amount of freshly isolated mitochondria (µg/well), which were purified by <b>A</b> DC or <b>B</b> MB methods. Briefly, succinate was used as a substrate to feed electrons to complex II and basal (state II) respiration was measured. State III respiration was determined in the presence of ADP. Oligomycin (Oligo) addition blocked ATP synthase and so evoked state IVo respiration. Maximum respiration (state uncoupled) was measured in the presence of the uncoupler FCCP. Finally, Antimycin A (Antim) was given to inhibit complex III. Data are represented as means of three independent experiments (5–8 replicates per groups) ± SD.</p