er respiratory activity and higher purity compared to the conventional DC method. A New Method to Isolate Functional Mitochondria operator. In addition, this method is suitable for the automated homogenization of multiple samples simultaneously. We tested the homogenization steps for different tissues and the integrity of the mitochondrial membranes was assessed by transmission EM analysis, oxygen consumption measurements and determination of the respiratory control ratio. Transmission EM analysis revealed homogenous mitochondrial fractions with a good membrane integrity and purity isolated by anti-TOM22 magnetic beads from different mouse tissues. Our Western blot and Clark electrode assays confirmed the high purity and functionality of the mitochondria. Notably, mitochondrial isolated either from soft or hard tissues showed reliable functionality. In addition, we compared the purity and oxygen consumption capacity of liver mitochondria isolated with the usual differential Saracatinib cost centrifugation or magnetic beads methods. Our proteomics, Western blot and XF Extracellular Flux Analyzer analyses indicated that the MB liver mitochondrial fraction contained more mitochondrial and less non-mitochondrial contaminants. Moreover, most likely due to this increased purity, they also showed elevated oxygen consumption capacity compared to DC isolated mitochondria. On the other hand DC isolated liver mitochondria showed higher A New Method to Isolate Functional Mitochondria RCR, which could indicate that MB purified mitochondria are slightly more leaky for hydrogen ions but according to our experience the obtained 3.5 ratio still represents a good membrane integrity. Since the traditional Clark electrode can only measure one sample simultaneously and mitochondrial measurements in this system need continuous manipulations, a new automated, highthroughput technique has been introduced. With the 
XF Extracellular Flux Analyzer one can measure 24 or 96 samples 20573509 in parallel. However, such measurements with high sample numbers require a reproducible technique for isolating pure mitochondria simultaneously from multiple tissue samples, which is less work intensive than the differential centrifugation protocol. Since mitochondria isolated from tissues by the combination of methods described here showed a reliable quality and quantity and these methods allow easy handling of numerous biological samples simultaneously, we propose that our approach will be valuable for many high-throughput downstream applications. calculated from 7473193 protein abundances between MB and DC fractions and are represented as a linear ratio. When these ratios were less than 1 the negative inverse ratio was taken and the values were transposed by the equation: 21/. All ratios are also depicted in Fig. 4BD and the identified protein names are shown in this table. Numbers indicate the order of the proteins in Fig. 4BD from left to right. Statistical analyzes were made by Mann-Whitney U test and p values were corrected to multiple testing according to Benjamini&Hochberg. A false discovery rate ,10% was set as the significance level and significant proteins are denoted by stars, ns: no significance. Bone is a dynamic tissue that undergoes resorption and formation. Osteoclast -mediated bone resorption is crucial for normal bone homeostasis, and also plays a causative role in osteoporosis, rheumatoid arthritis, Paget disease, multiple myeloma, and bone metastasis of breast cancers. OCs, which are ess