These data are similar to those detected in previous studies by other authors

asurement of cell index in each well. Cell index represents a quantitative measure of cell number present in a well e.g. low cell index reflects fewer cells attached to the electrodes. Approximately 10 x 103 of MEFs were plated into E-plate 16 and doubling time of cells were analysed based on real time measurements carried out for 48 hours. buy K 858 Cellular respiration Cellular respiration was measured polarographically, with the use of Oxygraph-2k. Cells from 10 cm diameter tissue PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755563 culture dish were 2 / 18 Mitofusin, Mitochondria and Energy Metabolism in MEF Cells trypsinized and resuspended in pre-warmed PBS to final protein concentration 0.150.3 mg/ ml. Oxygen consumption was measured at 37C in substrate free solution and after administration of 1 mM pyruvate, 5 mM glucose, 0.1 mg/ml oligomycin and CCCP. After respiration assay, cell suspension was mixed with 0.5 M NaOH and protein concentration was measured. Data were presented as mean use of O2 picomoles per milligram of protein per second. Mitochondrial membrane potential Mitochondrial potential was measured fluorimetrically according to the Cossarizza et al. . Cells were trypsinized, incubated with 5 M JC-1 dye at 37C in the dark for 15 min. Parallel to JC-1 cell also were labeled with the nuclear stain TO-PRO-3. When indicated, prior to JC-1/ TO-PRO-3 staining, cells were preincubated for 10 min with 3 M valinomycin and 5 M CCCP or 0.1 g/ml oligomycin. Fluorescence was measured by BD FACSCalibur flow cytometry equipped with argon laser and excitation/emission 642/661 for TO-PRO-3. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755095 JC-1 fluorescence was measured only in TO-PRO-3 negative cells. Blue Native sample preparation, electrophoresis and in-gel complex V activity assay MEFwt and MEFMfn2-/- cells were trypsinized, washed with cold PBS and homogenized in homogenization buffer on ice. Homogenates were centrifuged at 1000 x g for 5 min. at 4C. Supernatants were spun at 10 000 x g for 20 min at 4C, to obtain pellet enriched in mitochondria. Samples containing 100 g protein were pelleted and resuspended in 10 l aminocaproic acid buffer with PMSF and protease inhibitor cocktail. 1 l of 10% dodecylomaltoside was added to each sample. After 30 min incubation on ice, samples were spun at 17 000 x g for 20 min at 4C. Supernatants were mixed with 0.4 l aminocaproic acid buffer with 5% Coomassie G-250 and loaded onto 512% gradient acrylamide gel. Electrophoresis was started with 70 V in cathode buffer and anode buffer. After 1 h, voltage was increased to 200 V. To visualize complex V activity, gel stripes were incubated over night at room temperature in solution containing 35 mM Tris HCl, 270 mM glycine, 14 mM MgCl2, 0.1% Pb 2, 8 mM ATP according to Sabar et al., 2005 and Yan et al., 2009. An intensity of bands corresponds to ATP-ase enzymatic activity. Western blot analysis of selected respiratory chain proteins and mitochondrial ATP synthase subunit were performed with MitoProfile Total OXPHOS Rodent WB Antibody Cocktail. Cell extracts were prepared with Lysis Buffer, separated by 12% SDS-polyacrylamide gel electrophoresis and electro-transferred to nitrocellulose membrane. In WB, following subunits were detected: NDUFB8, CII-30 kDa; CIII-Core 2 protein; C-IV-I subunit; C-V-alpha subunit. Data were expressed as relative immunoreactivity of each complex in knockout cells in relation to wild type one. To study the mitochondrial biogenesis the following primary antibodies were used: TFAM, TOM20, PGC-1 and PCNA, -actin and GAPDH as loading marke