Edit Abstract: Sobotka 2017 MiPschool Obergurgl

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Abstract:	[[Image:MITOEAGLE-logo.jpg\|left\|100px\|link=http://www.mitoglobal.org/index.php/MITOEAGLE\|COST Action MitoEAGLE]] Substrate uncoupler inhibitor titration (SUIT) protocols allow us to study mitochondrial metabolic pathways and the electron transport system in various physiological and pathological situations. To study mitochondrial respiration, one faces the challenge to obtain valid data for as many respiratory states as possible in one measurement and still have quality control. SUIT protocols give us this opportunity with recently developed Reference protocols 1&2 for a wide use in comparative mitochondrial physiology. During mitochondrial respiration the oxygen consumption proceeds in cytochrome c oxidase. This simplifies the situation in comparison to ROS production measurements. It is known that the superoxide can be generated on multiple sites over the electron transfer-pathway. Therefore during ROS measurement the question is not only what the rate of this ROS production is, but also its site. Considering this critical issue we face the challenge to specify the exact spot of ROS generation, which is completely different to situation during high-resolution respirometry. To resolve this challenge new approach to respiratory protocol formulation is necessary. In our presentation we want to review basic concepts, significant milestones and most successful methods in the history of ROS measurement. We want to present our data from simultaneous respiration and hydrogen peroxide assessment using the Amplex Ultra Red assay. Our goal is also to point out and discuss the missteps and pitfalls which could be run into during a measurement by this method. We present our data considering the best possible methodological approach, meaning the optimal media and standard operating procedures.
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MiP areas:	Respiration Instruments;methods mt-Biogenesis;mt-density mt-Structure;fission;fusion mt-Membrane mtDNA;mt-genetics nDNA;cell genetics Genetic knockout;overexpression Comparative MiP;environmental MiP Gender Developmental biology Exercise physiology;nutrition;life style mt-Medicine Patients Pharmacology;toxicology mt-Awareness
Pathological states:	Aging;senescence Alzheimer's Autism Cancer Cardiovascular COPD Diabetes Inherited Infectious Myopathy Neurodegenerative Obesity Parkinson's Sepsis Other
Stress conditions:	Cell death Cryopreservation Ischemia-reperfusion Permeability transition Oxidative stress;RONS Temperature Hypoxia Mitochondrial disease
Organism:	Human Pig Mouse Rat Guinea pig Bovines Horse Dog Rabbit Cat Other mammals Chicken Zebrafish Drosophila Artemia Caenorhabditis elegans Saccharomyces cerevisiae Birds Reptiles Amphibians Fishes Chelicerates Crustaceans Hexapods Myriapods Annelids Molluscs Nematodes Other invertebrates Fungi Plants Protists Eubacteria Archea
Tissues;cells:	Heart Skeletal muscle Nervous system Liver Kidney Lung;gill Islet cell;pancreas;thymus Endothelial;epithelial;mesothelial cell Blood cells Fat Genital Other cell lines CHO HEK HeLa Neuroblastoma Fibroblast HUVEC Macrophage-derived Lymphocyte Platelet Stem cells
Preparations:	Intact organism Intact organ Permeabilized cells Permeabilized tissue Homogenate Isolated mitochondria SMP Chloroplasts Enzyme Oxidase;biochemical oxidation Intact cells
Enzymes:	Adenine nucleotide translocase Complex I Complex II;succinate dehydrogenase Complex III Complex IV;cytochrome c oxidase Complex V;ATP synthase Inner mt-membrane transporter Marker enzyme Supercomplex TCA cycle and matrix dehydrogenases Uncoupling protein
Regulation:	Aerobic glycolysis ADP ATP ATP production AMP Calcium Coupling efficiency;uncoupling Cyt c Flux control Inhibitor Ion;substrate transport mt-Membrane potential Oxygen kinetics PCr;Cr pH Phosphate Redox state Substrate Temperature Threshold;excess capacity Uncoupler Fatty acid Amino acid Q-junction effect
Coupling:	LEAK ROUTINE OXPHOS ET
Pathways:	F N S Gp DQ CIV NS Other combinations ROX
High-resolution respirometry:	Oxygraph-2k TIP2k O2k-Fluorometer pH NO TPP Ca O2k-Spectrophotometer O2k-Manual O2k-Protocol Theory NextGen-O2k
Event:	A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4 E1 E2 Oral Poster P-flash Review
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Free text: Any additional information (e.g. affiliations of authors). == Affiliations and support == :::: Sobotka O(1), Komlodi T(2), Doerrier C(2), Gnaiger E(2) ::::#Dept Physiol, Fac Med Hradec Kralove, Charles Univ, Czech Republic ::::#Oroboros Instruments, Innsbruck, Austria :::::: Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 [[MitoEAGLE]].

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