Oroboros Procedures

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Oroboros Open Support

Oroboros Procedures


Oroboros Procedures


Oroboros Procedures explain various applications of the Oroboros with Demo experiments.

We aim at a fundamental introduction into high-resolution respirometry and the elements of mitochondrial physiology. This mosaic evolves to combine the elements into a picture of modern mitochondrial respiratory physiology, progressively including user-innovation emerging within the O2k-Network.

»Mitochondr Physiol Network and »MiPNet



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Oroboros Procedures
Oroboros Procedures
» MitoPathways
Pesta 2012 Methods Mol Biol Fig7A.jpg
» Oroboros Demo experiments
Pesta 2012 Methods Mol Biol Fig7A.jpg
» Oroboros Analysis
O2k-Procedures
» Chemicals and media
PBI-Shredder O2k-Set
» Mitochondrial preparations
O2k-Protocols
» Mitochondrial and marker-enzymes

Implement your quality control management with the MitoFit QCS, to diagnose technical problems fast and cost-effectively with instrumental quality control tests.

Oroboros Procedures - with selected chapters of the Oroboros Manual

ProtocolReferencePreparationTissue and cellMammal and modelLast update
MiPNet21.14 Reference sample HRR
O2k-Protocols
Development of a reference sample for high-resolution respirometry.
Intact cells
Permeabilized cells
HEKHuman2024-12-06
MiPNet09.12 Manual titrations of SUIT chemicals
O2k-Protocols
Manual titrations of SUIT chemicals with 2.0 mL and 0.5 mL chambers of the Oroboros Bioenergetics Analyzer.
2024-11-25
Donnelly 2024 Redox BiolDonnelly C, Komlódi T, Cecatto C, Cardoso LHD, Compagnion A-C, Matera A, Tavernari D, Campiche O, Paolicelli RC, Zanou N, Kayser B, Gnaiger E, Place N (2024) Functional hypoxia reduces mitochondrial calcium uptake. Redox Biol 71:103037. https://doi.org/10.1016/j.redox.2024.103037Intact organism
Isolated mitochondria
Intact cells
Heart
Skeletal muscle
Human
Mouse
2024
Baglivo 2024 BECBaglivo E, Cardoso LHD, Cecatto C, Gnaiger E (2024) Stability of mitochondrial respiration medium used in high-resolution respirometry with living and permeabilized cells. Bioenerg Commun 2024.8. https://doi.org/10.26124/bec.2024-0008Permeabilized cells
Intact cells
HEKHuman2024
Timon-Gomez 2024 MitoFitTimón-Gómez A, Doerrier C, Sumbalová Z, Garcia-Souza LF, Baglivo E, Cardoso LHD, Gnaiger E (2024) Analysis of mitochondrial respiratory pathway and coupling control by substrate-uncoupler-inhibitor titration reference protocols. MitoFit Preprints 2024.8. https://doi.org/10.26124/mitofit:2024-0008Permeabilized cells
Intact cells
Homogenate
Isolated mitochondria
HEK
Heart
Nervous system
Liver
Blood cells
Human
Mouse
2024
Leo 2024 MitoFitLeo E, Rychtarova L, Garcia-Souza LF, Åsander Frostner E, Elmér E, Gnaiger E (2024) High-resolution respirometry in a small-volume chamber. MitoFit Preprints 2024.4.v2. https://doi.org/10.26124/mitofit:2024-0004.v2 - Accepted on 2024-10-27 as Manuscript JoVE67442R2 for publication in JoVEPermeabilized cells
Homogenate
Isolated mitochondria
Intact cells
Heart
Nervous system
Blood cells
HUVEC
Platelet
Human
Mouse
2024
MiPNet11.05 imt-pti-pce
O2k-Protocols
Isolated mitochondria or permeabilized tissues and cells.
Permeabilized cells
Permeabilized tissue
Isolated mitochondria
2022-04-10
MiPNet22.10 MiR05-Kit
O2k-Manual
Mitochondrial Respiration Medium - MiR05-Kit.
2022-03-03
Baglivo 2022 MitoFit-QCBaglivo E, Cardoso LHD, Cecatto C, Gnaiger E (2022) Statistical analysis of instrumental reproducibility as internal quality control in high-resolution respirometry. https://doi.org/10.26124/mitofit:2022-0018.v22022-08-04 published in Bioenerg Commun 2022.8.HEK2022
Baglivo 2022 BECBaglivo E, Cardoso LHD, Cecatto C, Gnaiger E (2022) Statistical analysis of instrumental reproducibility as internal quality control in high-resolution respirometry. Bioenerg Commun 2022.8. https://doi.org/10.26124/bec:2022-0008HEK2022
MiPNet24.10 H2O2 flux analysis
O2k-Protocols
Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
2021-10-22
MiPNet26.10 MgG data analysis
O2k-Protocols
Measurement of mitochondrial ATP production using Magnesium Green: DL-Protocols and data analysis with DatLab 7.4
2021-09-16
MiPNet24.09 Data analysis of mt-membrane potential
O2k-Protocols
Data analysis of mt-membrane potential.
2021-09-16
MiPNet20.06 IsolationMouseHeart-mt
Oroboros Protocols
Laboratory protocol: isolation of mouse heart mitochondria.
Isolated mitochondriaHeartMouse2021-08-09
MiPNet17.05 O2k-Fluo LED2-Module
O2k-Manual
O2k-Fluo LED2-Module.
2021-06-22
Went 2021 MitoFit PBWent N, Di Marcello M, Gnaiger E (2021) Oxygen dependence of photosynthesis and light-enhanced dark respiration studied by High-Resolution PhotoRespirometry. MitoFit Preprints 2021.05. https://doi.org/10.26124/mitofit:2021-0005Intact cellsAlgae2021
Cardoso 2021 BEC MgGCardoso LHD, Doerrier C, Gnaiger E (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. Bioenerg Commun 2021.1. https://doi.org/10.26124/bec:2021-0001Isolated mitochondriaHeartMouse2021
Komlodi 2021 BEC AmR-O2Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004Intact cellsOther cell linesSaccharomyces cerevisiae2021
Komlodi 2021 BEC QKomlódi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. Bioenerg Commun 2021.3. https://doi.org/10.26124/bec:2021-0003Isolated mitochondriaHeart
Nervous system
Mouse2021
MiPNet21.17 BloodCellsIsolation
O2k-Protocols
O2k-Protocols: Isolation of blood cells for HRR.
Intact cells
Permeabilized cells
Blood cells
Platelet
Human2020-10-22
MiPNet24.08 Safranin Analysis Template
O2k-Protocols
Excel template for safranin data analysis.
2020-05-13
MiPNet17.04 CitrateSynthase
O2k-Protocols
Laboratory protocol: Citrate synthase a mitochondrial marker enzyme.
Permeabilized cells
Permeabilized tissue
Homogenate
Isolated mitochondria
Enzyme
2020-04-18
MiPNet24.06 Oxygen flux analysis - DatLab 7.4
O2k-Protocols
Oxygen flux analysis with DatLab 7.4.
2020-04-16
MiPNet25.14 TPP Analysis Template
O2k-Protocols
Excel template for TPP data analysis.
2020-##-##
BEC 2020.1 doi10.26124bec2020-0001.v1Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1Permeabilized cells
Permeabilized tissue
Homogenate
Isolated mitochondria
2020
Gnaiger 2020 BEC MitoPathwaysGnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002Permeabilized cells
Permeabilized tissue
Homogenate
Isolated mitochondria
Intact cells
Heart
Skeletal muscle
Fibroblast
Human
Mouse
2020
MiPNet21.16 DatLab 7 Innovations
About Oroboros
DatLab 7: innovations.
2019-09-23
MiPNet23.12 DL-Protocols help
O2k-Protocols
DatLab Protocols: Help DatLab 7.
2019-09-13
MiPNet22.16 DatLab 7 Innovations: DL-Protocols
O2k-Protocols
DatLab 7.4 Innovations: DL-Protocols
2019-09-13
MiPNet24.11 mtMP calculation
O2k-Protocols
Mitochondrial membrane potential calculation
2019-08-05
MiPNet18.06 Amplex-Yeast
O2k-Protocols
O2k-FluoRespirometry: Amplex UltraRed using freeze-dried baker’s yeast. » Update: Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. MitoFit Preprints 2021.10. doi:10.26124/mitofit:2021-0010
2019-06-24
MiPNet20.14 AmplexRed H2O2-production
O2k-Protocols
O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed.
Isolated mitochondriaHeartMouse2019-06-24
MiPNet20.13 Safranin mt-membranepotential
O2k-Protocols
O2k-FluoRespirometry: HRR and simultaneous determination of mt-membrane potential with safranin or TMRM.
HomogenateNervous systemMouse2019-06-24
MiPNet18.05 Amplex-Mouse-heart
O2k-Protocols
O2k-Fluorometry: HRR and H2O2 production in mouse cardiac tissue homogenate.
HomogenateHeartMouse2019-06-24
Naszai 2019 Sci RepNászai A, Terhes E, Kaszaki J, Boros M, Juhász L (2019) Ca(2+)N it be measured? Detection of extramitochondrial calcium movement with high-resolution FluoRespirometry. Sci Rep 9:19229.Isolated mitochondriaLiverRat2019
Long 2019 Methods Mol BiolLong Q, Huang L, Huang K, Yang Q (2019) Assessing mitochondrial bioenergetics in isolated mitochondria from mouse heart tissues using Oroboros Oxygraph-2k. Methods Mol Biol 1966:237-46.Isolated mitochondriaHeartMouse2019
MiPNet12.01 Suppl T-issue
O2k-Protocols contents
MitoPathways at the Q-junction: mouse skeletal muscle fibers.
Permeabilized tissueSkeletal muscleMouse2018-11-18
Doerrier 2018 Methods Mol BiolDoerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3Permeabilized cells
Permeabilized tissue
Homogenate
Isolated mitochondria
Intact cells
Heart
Skeletal muscle
Endothelial;epithelial;mesothelial cell
Blood cells
HEK
Platelet
Human
Mouse
Rat
Saccharomyces cerevisiae
2018
Komlodi 2018 Methods Mol BiolKomlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55. https://doi.org/10.1007/978-1-4939-7831-1_8Permeabilized cells
Permeabilized tissue
Skeletal muscle
HEK
Human
Mouse
2018
Lemieux 2017 Sci RepLemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840. doi:10.1038/s41598-017-02789-8Permeabilized tissueHeartMouse2017
MiPNet20.08 IsolationRatLiver-mt
O2k-Protocols
Laboratory Protocol: isolation of rat liver mitochondria.
Isolated mitochondriaLiverRat2016-11-18
MiPNet21.12 Comparison mt respiration media
O2k-Protocols
O2k-MultiSensor: Mitochondrial respiration media for HRR and simultaneous O2k-Fluorometry.
Intact cells
Permeabilized cells
HEKHuman2016-10-18
MiPNet21.13 Comparison mt-preparations
O2k-Protocols
O2k-Protocols: Mitochondrial preparations for HRR.
Permeabilized cells
Homogenate
Isolated mitochondria
HeartMouse2016-10-18
MiPNet03.02 Chemicals-MediaSelected media and chemicals for respirometry with mitochondrial preparations.
O2k-Protocols
2016-08-30
MiPNet08.09 CellRespiration
Oroboros Procedures
High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX.
Intact cellsBlood cells
Lymphocyte
Human2016-08-11
MiPNet10.04 CellRespiration
O2k-Protocols
An experiment with high-resolution respirometry: coupling control in cell respiration.
Intact cellsBlood cellsMouse2016-08-08
MiPNet19.18E O2 flux analysis - DatLab 6
Oroboros Manual
Oxygen flux analysis: DatLab real-time.
2016-08-08
MiPNet21.07 MitoFit DatLab PT
O2k-Protocols
MitoFit DatLab proficiency test.
Permeabilized cellsHuman2016-07-20
MiPNet20.07 IsolationRatBrain-mtLaboratory Protocol: isolation of rat brain mitochondria.
O2k-Protocols
Isolated mitochondriaNervous systemRat2016-06-18
MiPNet14.05 TPP-mtMembranePotential
O2k-Protocols contents
TPP+ and mt-membrane potential.
2016-04-01
MiPNet20.15 IsolationRatHeart-mt
O2k-Protocols
Laboratory protocol: isolation of rat heart mitochondria.
Isolated mitochondriaHeartRat2016-03-25
MiPNet17.15 PBI-Shredder Mouse-heart-brain-liver
Oroboros Protocols
Tissue homogenates for diagnosis of mitochondrial respiratory function: Mouse heart, brain and liver.
Homogenate2015-06-22
MiPNet14.14 PermeabilizedFiberPreparation
O2k-Protocols contents
Preparation of permeabilized muscle fibers for diagnosis of mitochondrial respiratory function.
Permeabilized tissueHeart
Skeletal muscle
Human2015-06-22
MiPNet17.02 PBI-Shredder manual
O2k-Manual
PBI-Shredder HRR-Set: preparation of tissue homogenates for diagnosis of mitochondrial respiratory function.
Homogenate2015-06-19
MiPNet17.03 Shredder vs Fibers
Oroboros Procedures
Mitochondrial respiration in permeabilized fibers versus homogenate from fish liver and heart. An application study with the PBI-Shredder.
HomogenateHeart
Liver
Fishes2015-06-18
MiPNet20.09 IsolationBeefHeart-mt
O2k-Protocols
Laboratory protocol: isolation of beef heart mitochondria.
Isolated mitochondriaHeartBovines2015-06-15
Gnaiger 2015 Scand J Med Sci SportsGnaiger E, Boushel R, Søndergaard H, Munch-Andersen T, Damsgaard R, Hagen C, Díez-Sánchez C, Ara I, Wright-Paradis C, Schrauwen P, Hesselink M, Calbet JAL, Christiansen M, Helge JW, Saltin B (2015) Mitochondrial coupling and capacity of oxidative phosphorylation in skeletal muscle of Inuit and caucasians in the arctic winter. Scand J Med Sci Sports 25 (Suppl 4):126–34. https://doi.org/10.1111/sms.12612Permeabilized tissueSkeletal muscleHuman2015
Makrecka-Kuka 2015 BiomoleculesMakrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. https://doi.org/10.3390/biom5031319Permeabilized cells
Homogenate
Isolated mitochondria
Intact cells
Heart
Nervous system
HEK
Human
Mouse
2015
Krumschnabel 2015 Methods Mol BiolKrumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. https://doi.org/10.1007/978-1-4939-2257-4_22Isolated mitochondriaNervous systemMouse2015
MiPNet12.11 MitoRespiration
O2k-Protocols
Flux control ratios in isolated mitochondria. OXPHOS capacity and respiratory control.
Isolated mitochondriaArtemia
Crustaceans
2014-02-20
Krumschnabel 2014 Methods EnzymolKrumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81. https://doi.org/10.1016/B978-0-12-416618-9.00009-1HomogenateNervous systemMouse2014
Van Bergen 2014 MitochondrionVan Bergen NJ, Blake RE, Crowston JG, Trounce IA (2014) Oxidative phosphorylation measurement in cell lines and tissues. Mitochondrion 15:24-33.Permeabilized cells
Isolated mitochondria
Intact cells
Nervous system
Blood cells
Human
Mouse
2014
Chinopoulos 2014 Methods EnzymolChinopoulos C, Kiss G, Kawamata H, Starkov AA (2014) Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption. Methods Enzymol 542:333-48. https://doi.org/10.1016/B978-0-12-416618-9.00017-0Permeabilized cellsHEKHuman2014
Elustondo 2014 Biochim Biophys ActaElustondo PA, Negoda A, Kane CL, Kane DA, Pavlov EV (2014) Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore. Biochim Biophys Acta 1847:231–40.Isolated mitochondriaLiverRat2014
Pesta 2012 Methods Mol BiolPesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibers from small biopsies of human muscle. Methods Mol Biol 810:25-58. https://doi.org/10.1007/978-1-61779-382-0_3Permeabilized cells
Permeabilized tissue
Intact cells
Skeletal muscle
Other cell lines
HEK
Fibroblast
HUVEC
Human2012
MiPNet08.12 IOC22
O2k-Protocols
NO effect on mitochondrial oxygen kinetics at low oxygen. O2k workshop Report.
Isolated mitochondriaLiverRat2011-12-11
MiPNet15.08 TPP electrode
O2k-Protocols
Ion selective electrode for TPP+ and high-resolution respirometry.
2011-12-11
Lemieux 2011 Int J Biochem Cell BiolLemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43:1729–38. https://doi.org/10.1016/j.biocel.2011.08.008Permeabilized tissueHeartHuman2011
MiPNet08.15 Complex-I
O2k-Protocols
Laboratory protocol: Complex I (NADH:Ubiquinone Oxidoreductase, EC 1.6.5.3). Mitochondrial membrane enzyme.
Enzyme2010-08-23
MiPNet08.18 LactateDehydrogenase
O2k-Protocols contents
Laboratory protocol: Lactate dehydrogenase. Cytosolic marker enzyme.
Enzyme2010-08-06
MiPNet08.13 mt-Isolation-RLM
O2k-Protocols
Laboratory protocol: Isolation of rat liver mitochondria.
Isolated mitochondriaLiverRat2010-08-03
Aguirre 2010 Biochim Biophys ActaAguirre E, Rodríguez-Juárez F, Bellelli A, Gnaiger E, Cadenas S (2010) Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells. Biochim Biophys Acta 1797:557-65. https://doi.org/10.1016/j.bbabio.2010.01.033Intact cells
Enzyme
Oxidase;biochemical oxidation
HEK2010
Gnaiger 2009 Int J Biochem Cell BiolGnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45. https://doi.org/10.1016/j.biocel.2009.03.013Permeabilized tissue
Isolated mitochondria
Skeletal muscleHuman
Mouse
2009
Gnaiger 2008 POSGnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52.Permeabilized cells
Permeabilized tissue
Intact cells
Blood cellsMouse2008
Gnaiger 2007 MitoPathways
O2k-Protocols
Gnaiger E ed (2007) Mitochondrial pathways and respiratory control. 1st ed. Oroboros MiPNet Publications, Innsbruck:96 pp. ISBN 978-3-9502399-0-4. — see 5th edition: Gnaiger 2020 BEC MitoPathways.
2007
Huetter 2004 Biochem JHütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919-28. https://doi.org/10.1042/BJ20040095Intact cellsFibroblastHuman2004
Gnaiger 2000 Life in the ColdGnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Berlin, Heidelberg:431-42. https://doi.org/10.1007/978-3-662-04162-8_45Permeabilized tissue
Isolated mitochondria
HeartRat2000
MiPNet04.05 Titration-Injection
O2k-Protocols contents
From step titration to ramp injection: Uncoupling by FCCP with TIP.
Intact cellsEndothelial;epithelial;mesothelial cell
HUVEC
Human1999-01
Steinlechner-Maran 1996 Am J Physiol Cell PhysiolSteinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol Cell Physiol 271:C2053-61. https://doi.org/10.1152/ajpcell.1996.271.6.C2053Intact cellsEndothelial;epithelial;mesothelial cell
HUVEC
Human1996
Template NextGen-O2k.jpg
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