x III activity was normalized to citrate synthase activity to take into account variations in the amount of mitochondrial and non-mitochondrial protein contamination between the samples and was given as CIII/CS ratio. Complex IV activity Cytochrome c oxidase activity was determined in intact isolated mitochondria using the Cytochrome c Oxidase Assay Kit. The colorimetric assay was based on the observation that a decrease in absorbance at 550 nm of ferrocytochrome c was caused by its oxidation to ferricytochrome c by cytochrome c oxidase. The Cytochrome c Oxidase Assay was performed as described previously. Complex IV activity was normalized to citrate synthase activity to take into account variations in the amount of mitochondrial and non-mitochondrial protein contamination between the samples and was given as CIV/CS ratio. Phase contrast microscopy and morphological analysis For the morphological analysis, cells were seeded at a density of 46104 cells/mL on coverslips previously coated with 0.05 mg/mL collagen. Phase contrast pictures were taken from living neuroblastoma cells using a Zeiss Axiolab microscope with a 406/1,2 W Korr objective equipped with a digital camera Zeiss AxioCam MRc. Preparation of isolated mitochondria for the determination of the activities of the single complexes I-IV Cells were incubated for 15 min in an ice-cold lysis buffer ). Then, cells were homogenized with a glass homogenizer, and the resulting homogenate was centrifuged at 800 g for 10 min at 4uC to remove MEK162 chemical information nuclei and tissue particles. The supernatant 1 
was saved and the 12600694 pellet resuspended in the lysis buffer. The homogenization step as well as the low-speed centrifugation step was repeated. The supernatant 2 was saved and added to S1. Combined mitochondriaenriched supernatants were centrifuged at 20,000 g for 15 min at 4uC to obtain the mitochondrial fraction. The Reactive oxygen species levels Cells were plated the day before at a density of 16105 cells/well in a 48 well plate. The mitochondria-associated ROS levels were determined using the fluorescent dye dihydrorhodamine at a concentration of 10 mM. The cells were loaded for 15 min with the dye. After washing twice with HBSS, the fluorescence was determined with the Victor2 reader multiplate at 485 nm /535 nm. Oxygen consumption of vital cells Mitochondrial oxygen consumption was measured at 37uC using an Oroboros Oxygraph-2k system. In contrast to the respiratory protocol measuring oxygen consumption of isolated mitochondria August 2010 | Volume 5 | Issue 8 | e12359 GBE Ameliorates OXPHOS in a rather artificial experimental environment, this protocol enables the determination of oxygen consumption in whole, vital cells under the most physiological conditions. Five millions of cells were added to 2 mL of a mitochondrial respiration medium containing 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2P04, 20 mM HEPES, 110 mM sucrose, 1g/l BSA. To measure 21885866 state 4 of complex I, 5 mM pyruvate and 2 mM malate were added and cells were permeabilised with 15 mg/mL digitonin. Afterwards, 2 mM ADP was added to measure state 3, and in order to increase the respiratory capacity, 10 mM glutamate was added. To study the effect of convergent complex I+II electron input on respiration, 10 mM of succinate was added. The integrity of mitochondrial membrane was checked through the addition of 10 mM cytochrome c. After determining coupled respiration, 0.4 mM FCCP phenyl-hydrazone) was added and re