O2k-Open Support

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OROBOROS INSTRUMENTS

OROBOROS support
        MitoPedia: O2k-Open Support         MitoPedia: O2k hardware         MitoPedia: DatLab         MitoPedia: SUIT         O2k-Procedures         O2k-Manual         O2k-Videosupport         O2k-Products        


O2k-Open Support

We provide service and support based on open innovation

O2k-Open Support



O2k-Open Support: technical help

Oroboros support system
O2k-Open Support includes Open Access pages that help to optimally use the O2k, to apply instrumental O2k-Quality Control, and perform user-service when needed.
  1. MitoPedia: O2k: find information quickly with MitoPedia, the Bioblast glossary with lists of key words and links to relevant pages. (1) O2k-Open Support Glossary, (2) MitoPedia: O2k hardware, (3) MitoPedia: DatLab
  2. O2k-Procedures: explain various applications of the O2k with O2k-Demo experiments
  3. O2k-Manual: topic specific manuals with step by step help
  4. O2k-Videosupport
  5. O2k-Products: is not merely a 'List of products', it presents a guided tour to the O2k and integrates the concept of Gentle Science and Open Science.



O2k-Quality Control

MitoFit-QCS
Many commonly raised issues of O2k-technical support are solved in the newest version of DatLab.
The O2 sensor and O2k chamber tests are important to eliminate instrumental artefacts (instrumental quality control, QC), distinguished from assay QC addressing problems of solution protocols (preparation of chemicals), sample preparation protocols (such as mitochondrial preparations), and SUIT protocols.
  1. Clean O2k-chambers and contamination-free incubation medium are required.
  2. Analysis of DatLab files recorded in instrumental QC tests is essential.
  3. Biological experiments are not suitable for trouble shooting.
The sequence of instrumental QC tests is strictly
  1. O2 sensor test
  2. O2k-chamber test
O2 sensor test

O2 sensor test

The OroboPOS (polarographic oxygen sensor) requires regular service at intervals - these may be more than 1 year. SOPs are available to determine if a sensor service is required at an earlier or later date:
» O2 sensor test: QC1: Oxygen sensor test

O2k-chamber test

If all quality control criteria of the O2 sensor test are met, the operator can be assured that the quality of the sensor signal is acceptable. Next, the quality of the O2k-chamber assembly has to be tested, described in detail:
» O2k-chamber test: QC2: Instrumental O2 background


Search for defective components: the 2-chamber design of the O2k

The two chamber design of the O2k has many benefits, including advantages in trouble shooting. By switching components between sides A and B of the O2k, it is usually possible to locate an instrumental problem, finding selectively a specific defective component of the O2k. This component can then be serviced by the end user or replaced by Oroboros without the need to ship the entire O2k.


O2k-Open Support by Oroboros experts

  • O2k-Open Support is given to individual users by Oroboros experts through our O2k-ticket system.
  • We welcome and value feedback. Technical and scientific questions, which are not yet addressed in the available guidelines or are difficult to trace on our website, are relevant to many specialists and beginners in the field.
  • O2k-Open Support aims at sharing our support communication openly with the scientific community, (1) improving the scope and documentation of the O2k-technology, and (2) providing Open Access educational examples on answering questions and finding solutions - in the spirit of Gentle Science.
  • O2k-Open Support by Oroboros experts does not replace the obligation to consult the O2k-Manual and relevant chapters on O2k-Procedures prior to contacting the O2k-support team.

O2k-Open Support agreement

  • O2k-Open Support aims at sharing our support communication openly with the scientific community.
We would like to publish your latest question and example files on our website. You can decide if your name, institutional and Email address are included.
Kindly have a look at http://wiki.oroboros.at/index.php/O2k-Open_Support#O2k-Open_Support_by_Oroboros_experts and give us your confirmation via return email.
Please note that regular consultancy fees might apply, should you not agree with our O2k-Open Support agreement.
We will be happy to assist you - in the spirit of Gentle Science ( http://www.bioblast.at/index.php/Gentle_Science ).
O2-Open Support















Repair

Hardware problems may require replacement of a defective spare part. Some electronic or mechanical defects may be solved only by repair of the O2k in the electronics workshop of Oroboros Instruments, e.g., a defective Peltier unit.
A cost estimation for repair of the O2k-Main Unit due to an electronic or mechanical defect can only be given after inspection of the O2k in our electronics workshop in Austria. Once a cost estimation has been provided, the repair is carried out with the customer’s consent. For shipment of the O2k, follow the detailed instructions.


O2k-Network

The O2k-Network

The Oroboros O2k-Network serves to connect and support: Contact for advice on applications of the O2k and high-resolution respirometry - the global network in mitochondrial physiology.
» The O2k-Network Labs: » O2k-Network
» O2k-Network discussion forum


Contact

» In case you need further technical support and for your valuable feedback, please contact support@oroboros.at
» Oroboros MitoFit Laboratory - open for innovation and cooperation.
» Oroboros Science Scholarships - are offered for focused research projects on mitochondrial physiology and high-resolution respirometry


MitoPedia: O2k-Open Support

TermAbbreviationDescription
Amplex UltraRedAmRAmplex UltraRed (AmR) is used as an extrinsic fluorophore for measurement of hydrogen peroxide production (ROS) by cells or mitochondrial preparations. The reaction of H2O2 and AmR is catalyzed by horseradish peroxidase to produce the red fluorescent compound resorufin (excitation wavelength 563 nm, emission 587 nm). The change of emitted fluorescence intensity is directly proportional to the concentration of H2O2 added, whereby the H2O2 is consumed.
Attached cellsMany cell types are grown in culter as attached cells, such as endothelial or neuronal cells in a monolayer.
Biological contaminationBiological contamination may be caused by microbial growth in and experimental chamber or in the experimental medium.
CalciumCaCa2+ is a major signaling molecule in both prokaryotes and eukaryotes. Its cytoplasmic concentration is tightly regulated by transporters in the plasma membrane and in the membranes of various organelles. For this purpose it is either extruded from the cell through exchangers and pumps or stored in organelles such as the endoplasmic reticulum and the mitochondria. Changes in the concentration of the cation regulate numerous enzymes including many involved in ATP utilizing and in ATP generating pathways and thus ultimately control metabolic activity of mitochondria and the of entire cell. Measuring changes in Ca2+ levels is thus of considerable interest in the context of high-resolution respirometry.
Calcium GreenCaGCalcium Green denotes a family of extrinsic fluorophores applied for measurement of Ca2+ concentration.
Closed chamberCThe O2k chamber can be used as a closed system or open system. Gas bubbles must be avoided.
DatLab 2DL2DatLab 2 (DL2) is a MS-DOS programe. DL2 is still used for analysis of oxygen kinetics, after exporting files recorded in recent DatLab versions. A user-friendly O2-kinetics module is in preparation (DL8).
Different O2 fluxes in left and right chamberWhat are potential causes for different O2 fluxes in the left and right chamber?
DithioniteDitZero oxygen solution powder, Na2S2O4, used for calibration of oxygen sensors at zero oxygen, or for stepwise reduction of oxygen concentration in instrumental O2 background tests.
Fatty acid oxidationFAOFatty acid oxidation (β-oxidation) is a multi-step process by which fatty acids are broken down to generate acetyl-CoA, NADH and FADH2 for further energy transformation. Fatty acids (short chain with 4–8, medium-chain with 6–12, long chain with 14-22 carbon atoms) are activated by fatty acyl-CoA synthases (thiokinases) in the cytosol. The mt-outer membrane enzyme carnitine palmitoyltransferase I (CPT 1) generates an acyl-carnitine intermediate for transport into the mt-matrix. Octanoate, but not palmitate, (eight- and 16-carbon saturated fatty acids) may pass the mt-membranes, but both are frequently supplied to mt-preparations in the activated form of octanoylcarnitine or palmitoylcarnitine. Electron-transferring flavoprotein complex (CETF) is located on the matrix face of the mt-inner membrane, and supplies electrons from fatty acid β-oxidation (FAO) to CoQ.
Filter-CapFilter-Cap: O2k-Fluo LED2-Module (O2k-Series D to G) sensors (Fluorescence-Sensor Green and Fluorescence-Sensor Blue) and O2k-FluoRespirometer (O2k-Series H) sensors (Smart Fluo-Sensor Green and Smart Fluo-Sensor Blue) are equipped with a removable Filter-Cap for exchange of optical filters for the optical pathways from the LED to the sample and from the sample to the photodiode.
Fluorescence-Control UnitFluorescence-Control Unit with O2k-Front Fixation, Current-Control (O2k-Chamber A and B) for regulation of light intensity of the LED in the fluorescence sensors. This item is a standard component of the O2k-Fluorescence LED2-Module.
GainThe gain is an amplification factor applied to an input signal to increase the output signal.
Hydrogen peroxideH2O2
Hydrogen peroxide
Hydrogen peroxide, H2O2 or dihydrogen dioxide, is one of several reactive oxygen intermediates generally referred to as reactive oxygen species (ROS). It is formed in various enzyme-catalyzed reactions (e.g., superoxide dismutase) with the potential to damage cellular molecules and structures. H2O2 is dismutated by catalase to water and oxygen. H2O2 is produced as a signaling molecule in aerobic metabolism and passes membranes more easily compared to other ROS.
MitoPedia: DatLab
MitoPedia: O2k hardware
MitoPedia: O2k-Open Support
MitoPedia: SUIT
Mitochondrial membrane potentialmtMP, Δψ [V]The mitochondrial membrane potential, mtMP, is the electric part of the protonmotive force, ΔpH+.

Δψ = ΔpH+ - ΔµH+ / F

mtMP or Δψ is the potential difference across the inner mitochondrial (mt) membrane, expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ electron or per electron moved across the transmembrane potential difference, with the number of ‘motive’ electrons expressed in the unit coulomb [C].
O2k repairO2k repair of defective hardware may require replacement of spare parts. Some electronic or mechanical defects may be solved only by repair of the O2k in the electronics workshop of Oroboros Instruments, e.g., a defective Peltier unit (temperature control).
O2k series
The serial number of each O2k is shown on a sticker at the rear of the O2k.
The O2k series is specified as the capital letter in the O2k serial number of the Oroboros O2k. A serial number G-#### or H-#### denotes an Oxygraph from the G or H series, while A-#### denotes an O2k from the A series. With DatLab running real-time connected to the O2k, the serial number of the currently connected O2k is displayed: (1) in the right corner of the status line, besides the DatLab version number (bottom), and (2) in windows O2k control [F7] and O2k configuration.
O2k-Chamber MitoPediaO2k-Chamber: 16 mm inner diameter, Duran glass polished, with standard operation volume (V) of 2 cm3 (2 mL). Two units of this item are standard components mounted in the main unit of the O2k-FluoRespirometer.
O2k-Fluo Smart-ModuleThe O2k-Fluo Smart-Module is a component of the O2k-FluoRespirometer(O2k-Series H). It is an amperometric add-on module to the O2k-Respirometer, adding a new dimension to high-resolution respirometry. Optical sensors are inserted through the front window of the O2k-glass chambers, for measurement of hydrogen peroxide production (Amplex UltraRed), ATP production (Magnesium green), mt-membrane potential (Safranin, TMRM), Ca2+ (Calcium green), and numerous other applications open for O2k-user innovation. Current DatLab version is included in the delivery of this module.
O2k-Open Support
O2k-Open Support agreementO2k-Open Support aims at providing expert help quickly. Please, help us sharing our support communication openly with the scientific community.
O2k-Peltier Temperature ControlO2k-Peltier Temperature Control: Built-in electronic thermostat controlling temperature for two O2k-Chambers in the range of 4 to 47 °C; ±0.002 °C (at room temperature). Continuous recording of the O2k-Copper Block temperature with DatLab. Temperature change from 20 to 30 °C within 15 min; cooling from 30 to 20 °C within 20 min. Integral component of the O2k-Main Unit. The electronic temperature control of the O2k replaced the conventional water jacket.
O2k-Products
O2k-Window FrameO2k-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. Two units of this item are standard components of the (Oroboros O2k-Core (O2k-Series D - G) ), mounted on the O2k-Main Unit.
O2k-pH ISE-ModuleO2k-pH ISE-Module: two pH electrodes and reference electrodes and accessories, two black PEEK stoppers, supported by the Oroboros O2k. It also includes the newest DatLab version. Quality control: Stability of Oroboros pH electrodes is tested for drift <15 µpH·s-1·mL-1.
Open chamberOThe term "open O2k-Chamber" refers to a situation in which the liquid phase is allowed to equilibrate with a gas phase, but the stopper is partially inserted using the Stopper-Spacer.
OroboPOS-ConnectorOroboPOS-Connector (blue POM), with male connection to OroboPOS sensor head (POS) and with cable and male plug fitting into O2k-Main Unit. Two units of this item are standard components of the O2k-Assembly Kit (O2k-FluoRespirometer), mounted on the OroboPOS-Holders at the O2k-Main Unit.
Oxygen calibration - DatLabO2 calibration is the calibration in DatLab of the oxygen sensor. It is a prerequisite for obtaining accurate measurements of respiration. Accurate calibration of the oxygen sensor depends on (1) equilibration of the incubation medium with air oxygen partial pressure at the temperature defined by the experimenter; (2) zero oxygen calibration; (3) high stability of the POS signal tested for sufficiently long periods of time; (4) linearity of signal output with oxygen pressure in the range between oxygen saturation and zero oxygen pressure; and (5) accurate oxygen solubility for aqueous solutions for the conversion of partial oxygen pressure into oxygen concentration. The standard oxygen calibration procedure is described below for high-resolution respirometry with the automatic calibration routine by DatLab.^
Oxygen flux - instrumental backgroundJ°O2Instrumental background oxygen flux, J°O2, in a respirometer is due to oxygen consumption by the POS, and oxygen diffusion into or out of the aqueous medium in the O2k-Chamber. It is a property of the instrumental system, measured in the range of experimental oxygen levels by a standardized instrumental background test. The oxygen regime from air saturation towards zero oxygen is applied generally in experiments with isolated mitochondria and living or permeabilized cells. To overcome oxygen diffusion limitation in permeabilized fibers and homogenates, an elevated oxygen regime is applied, requiring instrumental background test in the same range of elevated oxygen.
Oxygen kineticsOxygen kinetics describes the dependence of respiration of isolated mitochondria or cells on oxygen partial pressure. Frequently, a strictly hyperbolic kinetics is observed, with two parameters, the oxygen pressure at half-maximum flux, p50, and maximum flux, Jmax. The p50 is in the range of 0.2 to 0.8 kPa for cytochrome c oxidase, isolated mitochondria and small cells, strongly dependent on Jmax and coupling state.
Oxygen sensor testPOS testThe O2 sensor test is an important component of the MitoFit Quality Control System. The OroboPOS sensor test is described in detail in MiPNet06.03 POS-calibration-SOP, is performed after switching on the Oroboros O2k, and is required as a basis of technical service of the instrument.
Oxygen signalThe oxygen signal of the Oroboros O2k is transmitted from the electrochemical polarographic oxygen sensor (OroboPOS) for each of the two O2k chambers to DatLab. The primary signal is a current [mAmp] which is converted into a voltage [V] (raw signal), and calibrated in SI units for amount of substrance concentration [µmol·L-1 or µM].
PH calibration bufferspH calibration buffers are prepared to obtain two or more defined pH values for calibration of pH electrodes and pH indicator dyes.
Polarization voltageUA polarization voltage of 600 mV to 800 mV is applied between anode and cathode of the polarographic oxygen sensor, resulting in a current when oxygen is consumed. The current is converted by the electronics to a voltage (raw signal) which must not be confused with the polarization voltage.
Polarographic oxygen sensorPOSPolarographic oxygen sensors (POS) are operated with a polarization voltage between the cathode and anode, connected by an electrolyte. Cathode, anode and electrolyte are separated from the analyte by an oxygen-permeable membrane. Oxygen is reduced at the cathode such that the local oxygen concentration is maintained at zero, and diffuses along the concentration gradient from the stirred medium to the cathode, resulting in a linear calibration between oxygen partial pressure and electric current [Amp] (amperometric mode of operation). The OroboPOS is the POS applied in the Oroboros O2k.
Raw signal of the oxygen sensorRThe raw signal of the polarographic oxygen sensor is the voltage obtained after a current-to-voltage conversion in the O2k. O2k-Support page: Oxygen signal
SUITbrowserUse the SUITbrowser to find the best substrate-uncoupler-inhibitor-titration (SUIT) protocol for your research questions. Open the SUITbrowser: http://suitbrowser.oroboros.at/
SafraninSafSafranin is one of the most established dyes for measuring mitochondrial membrane potential by fluorometry. It is an extrinsic fluorophore with an excitation wavelength of 495 nm and emission wavelength of 587 nm. Safranin is a potent inhibitor of N-linked respiration and of the phosphorylation system. Synonyms: Safranin O, Safranin Y, Saranin T, Gossypimine, Cotton Red, Basic Red2
Search for defective O2k componentsThe 2-chamber design of the O2k helps to search for defective O2k components, by switching components linked to O2k chambers A and B between sides A and B.
Setting the oxygen concentration
Shipping an O2kFor shipping an O2k, standard operating procedures have to be followed to avoid damage of the instrument and unexpected delays. The O2k-Main Unit must be shipped only in Packing\O2k-Box 1, without O2k-Chambers and without OroboPOS. Two O2k-Chamber Holders, two OroboPOS-Holders and two OroboPOS-Connectors are attached to the O2k-Main Unit for transport.
Stopper\black PEEK\conical Shaft\central PortStopper\black PEEK\conical Shaft\central Port: with conical shaft and one central capillary (with PTFE, graphite, carbon fiber), Volume-Calibration Ring (A or B) for volume adjustment 1.5 to 3.2 ml; 2 mounted O-rings, with 8 spare O-rings (O-ring\Viton\12.5x1 mm). The black PEEK stoppers are required for optical O2k-MultiSensor Modules (O2k-Fluo_LED2-Module). Two units of this item are standard components of the O2k-Assembly Kit.
TIP2k syringe blockedWhen the TIP2k syringe is blocked, it must not be used with the TIP2k, and specific cleaning instructions should be followed.
TIP2k-ModuleTIP2k-Module - Titration-Injection microPump (TIP2k) for two-channel operation with the O2k-FluoRespirometer with automatic control by DatLab of programmable titration regimes and feedback control (oxystat, pH-stat). The TIP2k-Module includes TIP2k-Accessories: 4 Microinjection syringes, 1 Pkg. TIP2k-Needle Spacers with mounting tool, 1 TIP2k-Needle Safety Support and 1 Pkg. TIP2k Filter Papers, and the newest DatLab version.
TMRMTMRMTMRM (tetramethylrhodamine methyl ester) is an extrinsic fluorophore used as a probe to determine changes in mitochondrial membrane potential. TMRM is a lipophilic cation that is accumulated in the mitochondrial matrix in proportion to Δψmt. Upon accumulation of the dye it exhibits a red shift in its absorption and fluorescence emission spectrum. The fluorescence intensity is quenched when the dye is accumulated in the mitochondrial matrix.
Temperature plot emptyProblem: Layout "01 Calibration Exp. Gr3-Temp" is selected, a third plot is displayed on the screen but it remains empty (no plot is shown). Newer versions of DatLab include pre-installed layouts which do not recognize some channel designations from older O2k series.
TetraphenylphosphoniumTPP+Tetraphenylphosphonium (TPP+). A lipophilic molecular probe in conjunction with an ion selective electrode (ISE) for measuring the mitochondrial membrane potential.
Time resolutionTime resolution in respirometric measurements is influenced by three parameters: the response time of the POS, the data sampling interval and the number of points used for flux calculation.
Uninterrupted power supplyUPSA back-up power supply may be required to secure uninterrupted power supply.
Voltage (raw signal) of the oxygen sensorRThe raw signal of the polarographic oxygen sensor is the voltage obtained after a current-to-voltage conversion in the O2k. O2k-Support page: Oxygen signal
Volume-Calibration RingVolume-Calibration Ring (A or B) PVDF for O2k-Stoppers, white; attached to the stoppers.
Volume-Calibration Ring sVVolume-Calibration Ring sV (A or B), white PVDF, attached to PEEK O2k-Stopper sV.
Zero calibrationR0Zero calibration is together with air calibration one of the two steps of the OroboPOS calibration. It is performed in the closed chamber after all the oxygen has been removed by the addition of dithionite or by respiration of mitochondria or cells. Any incubation medium can be used for zero calibration with dithionite or sample. Unlike air calibration it is not necessary to perform a zero calibration each day.
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