MitoPedia: O2k-Open Support

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MitoPedia: O2k-Open Support

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O2k-Open Support

MitoPedia O2k and high-resolution respirometry: O2k-Open Support 

MitoPedia: O2k-Open Support list Frequently Asked Questions and keywords for help.
More details - »MitoPedia: O2k hardware« - »MitoPedia: DatLab« - »O2k-Open Support alert«

ATPTAdenosine triphosphate is a nucleotid and functions as the major carrier of chemical energy in the cells. As it transfers its energy to other molecules, it looses its terminal phosphate group and becomes adenosine diphosphate (ADP).
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 fluorescenet product according to the supplier is called UltroxRed in the case of Amplex UltraRed which has a very similar structure to resorufin). 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 the O2k-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).
DatLab oxygen flux: performance and data analysisThe quality of the results are strongly affected by the performance and data analysis. Therefore, we provide guidelines for performing and evaluating respirometric assays.
Different O2 fluxes in left and right chamberWhat are potential causes for different O2 fluxes in the left and right chamber?
DithioniteDithDithionite Na2S2O4 is the 'zero oxygen solution powder' used for calibration of oxygen sensors at zero oxygen, or for stepwise reduction of oxygen concentrations in instrumental O2 background tests. It is not recommended to use dithionite in experiments with biological samples or several multisensor approaches, for these see Setting the oxygen concentration.
Enable DL-Protocol editingIn DatLab 7.4, it is possible to edit a DL-Protocol and save it as DLPU. To enable it, select in the menu 'Protocols' the option 'Enable DL-Protocol editing', then select the plot in which the marks will be set (e.g., O2 flux per V). Select the 'Overview' window, where you will be able to edit events and marks names, definition/state, final concentration and titration volumes, as well as select a mark as 'multi' for multiple titration steps, skip a mark, or add a new event or mark. After saving, export a DL-Protocol User (DLPU) and load it before running the next experiments.

For more information:

PlayVideo.jpg Export DL-Protocol User (*.DLPU)
Export DL-Protocol User (*.DLPU)Export DL-Protocol User (*.DLPU) Protocol possess unique D### codes and comprise a fixed sequence of events and marks which cannot be changed by the user. However, the user may edit concentrations and titration volumes of injections and store the modified protocol as user-specific DL-Protocol [File]\Export\DL-Protocol User (*.DLPU).

If users of older DatLab versions than DatLab 7.4 wish to alter the nature of the chemicals used or the sequence of injections, we ask them to contact the O2k-Technical Support.

DatLab 7.4

DatLab 7.4 offers a new feature in DL-Protocols: flexibility. Fixed sequence of events and marks can be changed (Skip/Added) in a SUIT protocol by the user. Moreover, editions of text, instructions, concentrations and titration volumes of injections in a specific DL-Protocol can be edited and saved as user-specific DL-Protocol [File]\Export\DL-Protocol User (*.DLPU).

For more information, see: Enable DL-Protocol editing
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.
IlluminationF10The chambers of the Oroboros O2k are illuminated by an internal LED. The illumination is switched on and off in DatLab during the experiment by pressing [F10]. This illumination must be distinguished from light introduced into the chambers by LEDs for the purpose of spectrophotometric and fluorometric measurements. For these, the internal illumination must be switched off.
Living cellsvceCell viability in living cells should be >95 % for various experimental investigations, including cell respirometry. Viable cells (vce) are characterized by an intact plasma membrane barrier function. The total cell count (Nce) is the sum of viable cells (Nvce) and dead cells (Ndce). In contrast, the plasma membrane can be permeabilized selectively by mild detergents (digitonin), to obtain the mt-preparation of permeabilized cells used for cell ergometry. Living cells are frequently labelled as intact cells in the sense of the total cell count, but intact may suggest the alternative meaning of viable or unaffected by a disease or mitochondrial injury.
Magnesium GreenMgGMagnesium Green (MgG) belongs to the extrinsic fluorophores applied for measurement of mitochondrial ATP production with mitochondrial preparations. This dye fluoresces when bound to Mg2+. The technique to measure mitochondrial ATP production is based on the fact that Mg2+ present different dissociation constants for ADP and ATP, and the adenine nucleotide translocase (ANT) exchanges ATP for ADP.
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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 mL (2 cm3). The O2k-sV-Module has a small chamber volume of 0.5 mL.
O2k-Fluo Smart-Module
The 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.
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-OroboPOS-Holder MitoPediaThe OroboPOS-Holder (POS-Holder), made from blue POM, is screwed into the copper block of the O2k-Main Unit, guiding the OroboPOS-Connector with the OroboPOS sensor head (POS) to the O2k-Chamber, and keeping the OroboPOS-Connector in a fixed position for sealing the O2k-Chamber with the OroboPOS-Seal Tip. In addition, the OroboPOS-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.
O2k-Peltier MitopediaO2k-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-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-Module
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O2k-pH ISE-Module: two pH electrodes and reference electrodes and accessories
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-Connector MitoPediaOroboPOS-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 using instrumental calibraiton DL-Protocols in 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 [µA] which is converted into a voltage [V] (raw signal), and calibrated in SI units for amount of substrance concentration [µmol·L-1 or µM].
PBI-Shredder MitoPediaPBI-Shredder: Auxiliary O2k-Tool for tissue homogenate preparation with high mitochondrial yield and function.
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.
Proton fluxJH+Volume-specific proton flux is measured in a closed system as the time derivative of proton concentration, expressed in units [pmol·s-1·mL-1]. Proton flux can be measured in an open system at steady state, when any acidification of the medium is compensated by external supply of an equivalent amount of base. The extracellular acidification rate (ECAR) is the change of pH in the incubation medium over time, which is zero at steady state. Volume-specific proton flux is comparable to volume-specific oxygen flux [pmol·s-1·mL-1], which is the (negative) time derivative of oxygen concentration measured in a closed system, corrected for instrumental and chemical background. pH is the negative logarithm of proton activity. Therefore, ECAR is of interest in relation to acidification issues in the incubation buffer or culture medium. The physiologically relevant metabolic proton flux, however, must not be confused with ECAR.
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:
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 or parts, 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-Module MitoPediaTIP2k-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).
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.
Uncoupler titrationsIn uncoupler titrations various uncouplers, such as CCCP, FCCP or DNP are applied to uncouple mitochondrial electron transfer from phosphorylation (ATP synthase, ANT and phosphate transporter), particularly with the aim to measure ET capacity. ET capacity is maximum oxygen flux measured as noncoupled respiration with optimum uncoupler concentration.
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 imt 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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