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O2k-Fluorometer Series G - Former Series (up to 2017-July) - the experimental system complete for high-resolution respirometry (HRR) combined with fluorometry. The O2k-Fluorometer includes the O2k-Core, O2k-Fluo LED2-Module and TIP2k, and supports all other add-on O2k-Modules of the Oroboros O2k.

The O2k is a sole source apparatus with no other instruments meeting its test experiments on O2k-Specifications.

The O2k-Main Basic is an integral element of the O2k-Main Unit. The Oroboros O2k Main Basic has the following components:

• Stainless-Steel Housing
• Switching power supply
• Microprocessor for integrated control, A/D converters and data handling
• Copper-Block with windows to 2 O2k-Chambers
• 2 Amperometric OroboPOS Plugs
• TIP2k socket, providing the basis for add-on of the TIP2k
• 2 Potentiometric Plugs for ion sensitive electrodes (ISE: TPP+, Ca2+; pH), providing the basis for add-on of the O2k-MultiSensor Modules
• 2 Amperometric Plugs, providing the basis for add-on of the O2k-Fluo LED2-Module or NO (H₂S) sensors.
• USB-Port for connection with DatLab (PC or laptop not included)

O2k-Main Power Cable\120 V\US-CA, USA and Canada (120 V).

O2k-Main Power Cable\230 V\Europe.

O2k-Network Reference Laboratories build a WorldWide network on high-resolution respirometry and mitochondrial physiology, the Oroboros O2k-Network.

O2k-TPP⁺ ISE-Module: Potentiometric ion-selective electrodes for measurement of mitochondrial membrane potential

The O2k-USB Flash Drive is a component of the Oroboros O2k containing: DatLab, O2k-Manual, O2k-Protocols, O2k-Publications, and info on O2k-Workshops.

O2k-Window Frame: blue POM, with thread for fixation on the O2k-Main Unit, to be removed only for rare cleaning purposes and for front fixation of the Fluorescence-Control Unit, using the O2k-Window Tool.

O2k-Chamber: Duran® glass polished, with standard operation volumes (V) of 2.0 mL or 0.5 mL (small chamber volume in the O2k-sV-Module, 12 mm inner diameter). The optical properties of Duran® allow application of fluorometric sensors (Duran® optical properties).

The O2k-sV-Module is the O2k small-volume module, comprised of two Duran® glass chambers of 12 mm inner diameter specifically developed to perform high-resolution respirometry with reduced amounts of biological sample, and all the components necessary for a smaller operation volume V of 0.5 mL. The current DatLab version is included in the delivery of this revolutionary module.

OctGM: Octanoylcarnitine & Glutamate & Malate.

MitoPathway control state: FN

SUIT protocols: SUIT-015, SUIT-016, SUIT-017

OctM: Octanoylcarnitine & Malate.

MitoPathway control state: F

SUIT protocols: SUIT-002, SUIT-015, SUIT-016, SUIT-017

Respiratory stimulation of the FAO-pathway, F, by fatty acid FA in the presence of malate M. Malate is a type N substrate (N), required for the F-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the F-pathway capacity is overestimated, if there is an added contribution of NADH-linked respiration, F(N) (see SUIT-002). The FA concentration has to be optimized to saturate the FAO-pathway, without inhibiting or uncoupling respiration. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway. High concentration of malate, typically 2 mM, saturates the N-pathway.

OctPGMS: Octanoylcarnitine & Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control state: FNS

SUIT protocol: SUIT-001, SUIT-002, SUIT-015

This substrate combination supports convergent electron flow to the Q-junction.

OctPM: Octanoylcarnitine & Pyruvate & Malate.

MitoPathway control state: FN

SUIT protocol: SUIT-002, SUIT-005

This substrate combination supports N-linked flux which is typically higher than FAO capacity (F/FN<0 in the OXPHOS state). In SUIT-RP1, PMOct is induced after PM(E), to evaluate any additive effect of adding Oct. In SUIT-RP2, FAO OXPHOS capacity is measured first, testing for the effect of increasing malate concentration (compare malate-anaplerotic pathway control state, M alone), and pyruvate is added to compare FAO as the background state with FN as the reference state.

Open Access (OA) academic articles comprise all different forms of published research that are distributed online, free of charge and with an open license to facilitate the distribution and reuse. The open access repositories serve as the perfect vehicle to transmit free knowledge, including but not limited to peer-reviewed and non-peer-reviewed academic journal articles, conference papers, theses, book chapters and monographs. Driven by the problems of social inequality caused by restricting access to academic research, the Open Access movement changes the funding system of published literature allowing for more readers and thus increased access to scientific knowledge, as well as addressing the economic challenges and unsustainability of academic publishing. In addition to being free to read (gratis), open access articles may also be free to use (libre) where the copyright is held by the authors and not the publisher.

Definition by the Directory of Open Access Journals (DOAJ): "We define these as journals where the copyright holder of a scholarly work grants usage rights to others using an open license (Creative Commons or equivalent) allowing for immediate free access to the work and permitting any user to read, download, copy, distribute, print, search, or link to the full texts of articles, crawl them for indexing, pass them as data to software, or use them for any other lawful purpose."

The OroboPOS is a polarographic oxygen sensor (POS), with an amperometric mode of operation. The OroboPOS meets the highest quality criteria in terms of linearity, stability and sensitivity of the signal. The Clark type polarographic oxygen sensor (POS) remains the gold standard for measuring dissolved oxygen in biomedical, environmental and industrial applications over a wide dynamic oxygen range.

It consists of a gold cathode, a silver/silverchloride anode and a KCl electrolyte reservoir separated from the sample by a 25 µm membrane (FEP). The main body of the OroboPOS is made of PEEK. With application of a polarization voltage (0.8 V), a current is obtained as an amperometric signal, which is converted to a voltage.

Oroboros O2k-Core - Former Series (O2k-Series D - G) - the experimental system complete for basic high-resolution respirometry (HRR). The O2k-Core includes the O2k-Main Unit with stainless steel housing, O2k-Assembly Kit, two OroboPOS (polarographic oxygen sensors) and OroboPOS-Service Kit, DatLab software, the ISS-Integrated Suction System and the O2k-Titration Set. The O2k-Core supports all add-on O2k-Modules of the O2k.

On-line display of oxygen flux (rate of respiration) is provided in addition to the conventional 'oxygraphic' plot of oxygen concentration over time. Highest signal stability minimizes the required amounts of biological sample, and provides the basis for resolution in the extreme low-oxygen range. Peltier temperature control provides a thermal stability at ±0.002 °C in the range of 4 °C to 47 °C at typical constant room temperature. Electronically controlled PVDF or PEEK stirrers are integrated in the two-chamber design of the O2k, and a barometric pressure transducer enables automatic oxygen calibrations implemented in the DatLab software.

The O2k is a sole source apparatus with no other instruments meeting its specifications.

An outlier-skewness index OSI is defined for evaluation of the distribution of data sets with outliers including separate clusters or skewness in relation to a normal distribution with equivalence of the average and median. The OSI is derived from Pearson’s coefficient of skewness 2:

Pearson 2 coefficient = 3 · (average-median)/SD

The outlier-skewness index OSI introduces the absolute value of the arithmetic mean, m = ABS(average + median)/2, for normalization:

OSI = (average-median)/(m + SD)

OSI = (average-median)/[ABS(average+median)/2 + SD]

At the limit of a zero value of m, the OSI equals the Pearson 2 coefficient (without the multiplication factor of 3). At high m with small standard deviation (SD), the OSI is effectively the difference between the average and the median normalized for m, (average-median)/m.

Overlay of plots is defined in DatLab as selection of graph layouts showing identical plots from the two O2k-chambers in each graph. Overlay of plots is selected in Graph layout. Superimposed traces of flux/flow from chambers A and B are then shown in Graph 1, and of concentration in chambers A and B in Graph 2.

There are basically two ways to superimpose traces recorded in different experiments: Export of the graphics via windows metafile or export of the data to e.g. a spreadsheet program.

If you export via wmf you also can manipulate the graphics but then usually the lines are broken up in different segments. This can be done in various programs like MS Word, Open Office Draw and even in MSPower Point, though this maybe is the worst program to do this. It is better to manipulate them in a proper program like OO Draw, convert it to an unchangeable picture and then import it to a presentation graphics. Anyway, when you import directly to Power point (or other programs), make sure not to import it as a "picture" but as a metafile. Also in some programs you might afterwards have to "break" it up, or accept a "conversion to a MS Draw object" or other similar linguistic inventions of the software gurus. For this option we suggest to do as much as possible directly in DatLab (setting colors, line widths, ..) using the options in "Plots"/"select plots" and "graph"/"options".

The “hardcore“ option is to export the data and import it into e.g. a spreadsheet program (MS Excel , OOCalc). It takes longer to have a simple overlay but gives you far less problems later and its easier to make changes later. To do this you can export your dataset "Export"/"Data to Textfile" and then go from there.

2-Oxoglutaric acid or alpha-ketoglutaric acid, C₅H₆O₅, occurs under physiological conditions as the anion 2-Oxoglutarate^2-, Og. 2-Oxoglutarate (alpha-ketoglutarate) is formed from isocitrate as a product of isocitrate dehydrogenase (IDH) in the TCA cycle, and is a substrate of oxoglutarate dehydrogenase (OgDH). The 2-oxoglutarate carrier exchanges malate^2- for 2-oxoglutarate^2- as part of the malate-aspartate shuttle. In the cytosol, oxoglutarate+aspartate are transaminated to form oxaloacetate+glutamate. Cytosolic malate dehydrogenase converts oxaloacetate+NADH to malate.

The PB-Module has been developed for conducting measurements of PhotoBiology, including photosynthesis. It consists of the PB Light Source and electronic components which are an integral part of the NextGen-O2k. Measurements are recorded and evaluated with the DatLab 8 software.

PBI-Shredder SG3 for tissue homogenate preparation, heavy duty high torque SG3 driver with convertible handle, SG3 base with 3 position force setting lever (FSL), battery charger and two lithium ion batteries. The PBI-Shredder SG3 is included in the PBI-Shredder O2k-Set. Select 230 V or 120 V.

Oroboros Instruments: world-wide distributor.

PGMS: Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control state: NS-pathway control state

2-oxoglutarate is produced through the citric acid cycle from citrate by isocitrate dehydrogenase, from oxaloacetate and glutamate by the transaminase, and from glutamate by the glutamate dehydrogenase. If the 2-oxoglutarate carrier does not outcompete these sources of 2-oxoglutarate, then the TCA cycle operates in full circle with external pyruvate&malate&glutamate&succinate

pH combination electrode, 150 mm shaft, 6 mm diameter, incl. connection cable with BNC plug. Discontinued

• Replaced by O2k-pH ISE-Module.

pH-Glass electrode with BNC plug.

PMS: Pyruvate & Malate & Succinate.

MitoPathway control: CI&II

Pyruvate (P) is oxidatively decarboxylated to acetyl-CoA and CO₂, yielding NADH catalyzed by pyruvate dehydrogenase. Malate (M) is oxidized to oxaloacetate by mt-malate dehydrogenase located in the mitochondrial matrix. Condensation of oxaloacate with acetyl-CoA yields citrate (citrate synthase). This documents an additive effect of convergent CI&II electron flow to the Q-junction, with consistent results obtained with permeabilized muscle fibres and isolated mitochondria (Gnaiger 2009).

The POS-Holder, made from blue POM, is screwed into the copper block of the O2k-Main Unit, guiding theguiding the POS to the 2-mL O2k-chamber, and keeping the SmartPOS/OroboPOS-Connector in a fixed position for sealing the O2k-chamber with the POS-Seal Tip. In addition, the POS-Holder fixes the O2k-Chamber in an accurate rotational position by pressing against the angular cut of the glass chamber.

Two units of this item are standard components mounted on the O2k-Main Unit.

POS-Membrane Ring, PEEK, holds the membrane against the inner O-ring on the POS housing.

POS-Mounting Tool for application of POS-Membranes. It consists of two parts, (i) the membrane guide (larger diameter) and (ii) the membrane ring holder with O-ring\Viton\6x1 mm for holding the POS-Membrane Ring during membrane application. Since the O2k-Series G-0075 membrane ring holder have a 2 mm extended ridge for a better grip.

Packing\Peli Case: Watertight, crushproof, and dust proof case for safe transportation of the O2k:

Retractable extension handle, Strong polyurethane wheels with stainless steel bearings, Easy open Double Throw latches, Open cell core with solid wall design - strong, light weight, O-ring seal, Automatic Pressure Equalization Valve, Fold down handles, Stainless steel hardware and padlock protectors, 4 level Pick N Pluck™ with convoluted lid foam, Special foam inlets for O2k.

Interior Dimensions: 21.48" x 16.42" x 12.54" (54.5 x 41.7 x 31.8 cm)

PalOctM: Palmitoylcarnitine & Octanoylcarnitine & Malate.

MitoPathway control state: Fatty acid oxidation pathway control state

SUIT protocols: SUIT-019

PalOctPGMS: Palmitoylcarnitine & Octanoylcarnitine & Pyruvate & Glutamate & Malate & Succinate.

MitoPathway control state: FNS

SUIT protocols: SUIT-019

PalPGMSGp: Palmitoylcarnitine & Pyruvate & Glutamate & Malate & Succinate & Glycerophosphate.

MitoPathway control state: FNSGp

SUIT protocol: SUIT-026

This substrate combination supports convergent electron flow to the Q-junction.

The pascal [Pa] is the SI unit for pressure. [Pa] = [J·m^-3] = [N·m^-2] = [m^-1·kg·s^-2].

The standard pressure is 100 kPa = 1 bar (10⁵ Pa; 1 kPa = 1000 Pa). Prior to 1982 the standard pressure has been defined as 101.325 kPa or 1 standard atmosphere (1 atm = 760 mmHg).

PeerJ Preprints is the 'pre-print' area of the Open Access journal PeerJ. Similar to preprint servers that already exist (for example arXiv.org), authors can submit draft, incomplete, or final versions of articles they are working on. By using this service, authors establish precedent; they can solicit feedback; and they can work on revisions of their manuscript. Once they are ready, they can submit their preprint article into PeerJ (although it is not a requirement to do so).

PeerJ Preprints was launched in April 2013. It only accepts submissions in the same subject areas as PeerJ (biological, medical and environmental sciences) and PeerJ Computer Science. In order to submit to PeerJ Preprints, at least the submitting author must have a user account with PeerJ. There is no pre-publication peer-review of submissions; however we do perform basic checks to ensure conformity with our policies. Submissions are made using the same platform as with the peer-reviewed journals, although some of the requirements are less stringent. Articles are not typeset, but we do provide automated conversion into PDF. The default is for a PeerJ Preprints publication to be fully open to all viewers (what we call a 'public' pre-print).

PeerJ is an Open Access, peer-reviewed, scholarly journal. It considers and publishes research articles in the biological, medical and environmental sciences. It aims for rapid decision making and will publish articles as soon as they are ready. PeerJ is based in both San Diego, US, and London, UK.

Permeabilized muscle fibers (pfi) are used as a mitochondrial preparation in respirometry to access mitochondrial function comparable to isolated mitochondria (imt). pfi are obtained by selectively permeabilizing the plasma membrane mechanically and chemically (saponin), for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes.

» MitoPedia topic: Mitochondrial preparations

Permeabilized tissue (pti, see also permeabilized muscle fibers, pfi) or cells (pce) are mitochondrial preparations obtained by selectively permeabilizing the plasma membrane mechanically or chemically, for the exchange of soluble molecules between the cytosolic phase and external medium, without damaging the mt-membranes.

Permeabilized cells (pce) are, therefore, not any longer viable or living cells (ce), since the intactness of cells implies the intactness of the plasma membrane. Any typical quantiative cell viability test (trypan blue etc) evaluating the intactness of the plasma membrane, yields a 100% negative result on fully permeabilized cells.

From Gnaiger 2014 MitoPathways

The phosphorylation pathway (phosphorylation system) is the functional unit utilizing the protonmotive force to phosphorylate ADP (D) to ATP (T), and may be defined more specifically as the P»-system. The P»-system consists of adenine nucleotide translocase, phosphate carrier, and ATP synthase. Mitochondrial adenylate kinase, mt-creatine kinase and mt-hexokinase constitute extended components of the P»-system, controlling local AMP and ADP concentrations and forming metabolic channels. Since substrate-level phosphorylation is involved in the TCA-cycle, the P»-system includes succinyl-CoA ligase (GDP to GTP or ADP to ATP).

Plunger\10 mm3 for Microsyringe\10 mm3 51/0.13 mm, spare.

Plunger\50 mm3 for Microsyringe\50 mm3 51/0.15 mm, spare. Discontinued

Polishing Cloth for cathode cleaning. Replace the Polishing Cloth at intervals. A two-year interval may be considered in cases of intensive use.

Power O2k Numbers: Single number to label the O2k in a Power O2k-Lab.

The Power O2k-Respirometer is an economical option for using additional O2k-Units to increase output in high-resolution respirometry.

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Proline (Pro), C₅H₉NO₂, is an amino acid which occurs under physiological conditions mainly in the nonpolar form, with pK_a1 = 1.99 pK_a2 = 10.96. Proline is an anaplerotic substrate that supports both the proline pathway control state and the glutamate-anaplerotic pathway control state. Proline is used as a single substrate or in combination with carbohydrate-derived metabolites in mitochondria particularly of flight muscle of many (but not all) insects. Proline is oxidized to delta-1-pyrroline-5-carboxylate by the mtIM L-proline:quinone oxidoreductase (proline dehydrogenase, ProDH), with reduction of FAD to FADH₂ and direct entry into the Q-junction. delta-1-pyrroline-5-carboxylate is converted to glutamate by 1-pyrroline-5-carboxylate dehydrogenase.

A prosthetic group is a cofactor that is attached permanently and tightly or even covalently to an enzyme and that is regenerated in each enzymatic turnover. Thus a prostethic group is distinguished from a coenzyme or cosubstrate that is attached loosely and transiently. Like a coenzyme, the prosthetic group is required by an enzyme for its activity. A prosthetic group is 'a tightly bound, specific nonpolypeptide unit in a protein determining and involved in its biological activity' (IUPAC definition).

FMN/FMNH₂ and FAD/FADH₂ are prosthetic groups of Complex I and Complex II, respectively.

The protonmotive force ∆_mF_H⁺ is known as Δp in Peter Mitchell’s chemiosmotic theory [1], which establishes the link between electric and chemical components of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the pmF ranks among the most fundamental theories in biology. As such, it provides the framework for developing a consistent theory and nomenclature for mitochondrial physiology and bioenergetics. The protonmotive force is not a vector force as defined in physics. This conflict is resolved by the generalized formulation of isomorphic, compartmental forces, ∆_trF, in energy (exergy) transformations [2]. Protonmotive means that there is a potential for the movement of protons, and force is a measure of the potential for motion.

The pmF is generated in oxidative phosphorylation by oxidation of reduced fuel substrates and reduction of O₂ to H₂O, driving the coupled proton translocation from the mt-matrix space across the mitochondrial inner membrane (mtIM) through the proton pumps of the electron transfer pathway (ETS), which are known as respiratory Complexes CI, CIII and CIV. ∆_mF_H⁺ consists of two partial isomorphic forces: (1) The chemical part, ∆_dF_H⁺, relates to the diffusion (d) of uncharged particles and contains the chemical potential difference^§ in H⁺, ∆µ_H⁺, which is proportional to the pH difference, ∆pH. (2) The electric part, ∆_elF_p⁺ (corresponding numerically to ∆Ψ)^§, is the electric potential difference^§, which is not specific for H⁺ and can, therefore, be measured by the distribution of any permeable cation equilibrating between the negative (matrix) and positive (external) compartment. Motion is relative and not absolute (Principle of Galilean Relativity); likewise there is no absolute potential, but isomorphic forces are stoichiometric potential differences^§.

The total motive force (motive = electric + chemical) is distinguished from the partial components by subscript ‘m’, ∆_mF_H⁺. Reading this symbol by starting with the proton, it can be seen as pmF, or the subscript m (motive) can be remembered by the name of Mitchell,

∆mFH+ = ∆dFH+ + ∆elFp+

With classical symbols, this equation contains the Faraday constant, F, multiplied implicitly by the charge number of the proton (z_H⁺ = 1), and has the form [1]

∆p = ∆µH+∙F-1 + ∆Ψ

A partial electric force of 0.2 V in the electrical format, ∆_elF_eH⁺a, is 19 kJ∙mol^-1 H⁺_a in the molar format, ∆_elF_np⁺a. For 1 unit of ∆pH, the partial chemical force changes by -5.9 kJ∙mol^-1 in the molar format, ∆_dF_nH⁺a, and by 0.06 V in the electrical format, ∆_dF_eH⁺a. Considering a driving force of -470 kJ∙mol^-1 O₂ for oxidation, the thermodynamic limit of the H⁺_a/O₂ ratio is reached at a value of 470/19 = 24, compared to the mechanistic stoichiometry of 20 for the N-pathway with three coupling sites.

The Q-Sensor has been designed as a part of the Q-Module for measurements with cyclic voltammetry and voltammetry, allowing for analysis of the Q redox state. The Q-Stopper with the reference electrode is called Q-Sensor, which is plugged in the NextGen-O2k. A three-electrode system is used to detect the Q redox state. Two of the three electrodes (glassy carbon and platinum electrode) are built into the Q-Stopper, while the reference electrode is removable (Reference-Electrode\2.4 mm).

The Q-junction is a junction for convergent electron flow in the Electron transfer pathway (ET-pathway) from type N substrates and mt-matrix dehydrogenases through Complex I (CI), from type F substrates and FA oxidation through electron-transferring flavoprotein Complex (CETF), from succinate (S) through Complex II (CII), from glycerophosphate (Gp) through glycerophosphate dehydrogenase Complex (CGpDH), from choline through choline dehydrogenase, from dihydro-orotate through dihydro-orotate dehydrogenase, and other enzyme Complexes into the Q-cycle (ubiquinol/ubiquinone), and further downstream to Complex III (CIII) and Complex IV (CIV). The concept of the Q-junction, with the N-junction and F-junction upstream, provides the rationale for defining Electron-transfer-pathway states and categories of SUIT protocols.

The Q-redox state reflects the redox status of the Q-junction in the mitochondrial or chloroplast electron transfer system (ETS). Coenzyme Q (CoQ or Q, ubiquinone) is a mobile redox component located centrally in the mitochondrial ETS, while plastoquinones are essential mobile components in the photosynthetic system with a similar function. The Q-redox state depends on the balance between reducing capacities of convergent electron entries from fuel substrates into the Q-junction and oxidative capacities downstream of Q to the electron acceptor oxygen. Therefore, deficiencies in the mitochondrial ETS, originating from e.g. the malfunction of respiratory Complexes, can be detected by measuring the changes of the Q-redox state with respect to the respiratory activity.

A three-electrode system was implemented into the NextGen-O2k to monitor the Q-redox state continuously and simultaneously with respiratory oxygen consumption. Added CoQ2 reflects the mitochondrial Q-redox state when equilibrating both with the detecting electrode and the biological sites (e.g. Complexes I, II and III).

RS232 0-modem-Cable for connecting the O2k-Main Unit to the PC or laptop with DatLab installed. When using DatLab 5, this cable is replaced by USB-Cable 2.0\Type A-B for O2k-Series E upwards.

Discontinued

Reference-Electrode\2.4 mm: 2.4 mm diameter glass shaft, for ISE.

Replacement-Barrel for Reference-Electrode\2.4 mm, 2.4 mm diameter glass.