Oroboros Instruments O2k Manual
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Oroboros Instruments O2k Manual

Multisensor system withion selective electrodes (ise)
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Oroboros O2k-Manual
Mitochondrial Physiology Network 15.03(08):1-26 (2018)
Version 08: 2018-01-25 ©2010-2018 Oroboros
Updates:
http://wiki.oroboros.at/index.php/MiPNet15.03_O2k-MultiSensor-ISE
O2k-MultiSensor
system with
ion selective
electrodes (ISE)
Fasching M, Gnaiger E
Oroboros Instruments
High-Resolution Respirometry
Schöpfstrasse 18, A-6020 Innsbruck, Austria
Email:
instruments@oroboros.at
www.oroboros.at
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Oroboros Instruments
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High-resolution respirometry
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Summary of Contents for Oroboros Instruments O2k

  • Page 1: Table Of Contents

    5.1. pX signal 5.2. Configuration and gain 5.3. Calibration Supplement A: DatLab 5.2. Determine the O2k series O2k series B and C, pX upgrade installed before 2011 O2k series B and C, pX upgrade installed after 2010 High-resolution respirometry Oroboros Instruments...

  • Page 2: Introduction And Scope

    MiPNet15.03 O2k-MultiSensor-ISE 1. Introduction and scope The pX channel of the Oroboros O2k yields a record of a potentiometric (voltage) signal simultaneously with the oxygen signal in both O2k- chambers. O2k-TPP ISE- Module consists of two ion-selective electrodes (ISE) separate reference electrodes.

  • Page 3: The Ion Selective Electrode (Ise) System

    2 Reference-Electrode\2.4 mm: 2.4 mm diameter glass shaft, for ISE 4 Replacement-Barrel for Reference-Electrode\2.4 mm (10) Electrolyte\Reference-Electrode Manual O2k-MultiSensor System with ISE For O2k Series B+C with pX upgrade installed before 2011 only (11) MultiSensor-Connector for separate reference electrode (12) Grounding cable with Allen key...

  • Page 4: Assembly Of The Ise

    MiPNet15.03 O2k-MultiSensor-ISE 2.2. Assembly of the ISE The ISE is delivered in an assembled state but without filling solution or membrane. Before its first use it must be disassembled. ISE-Membrane Holder, lower part of electrode housing ISE-Electrode Holder, middle part of electrode housing...
  • Page 5 MiPNet15.03 O2k-MultiSensor-ISE 2.2.2. Membrane mounting Use a good light source. Dry all plastic parts (especially the inside of parts A, E, and F) with a paper towel. 1. With forceps take membrane from membrane box and remove the paper covers on both sides of the membrane.
  • Page 6 MiPNet15.03 O2k-MultiSensor-ISE 7. Finally press E+F gently against membrane D with the flat end of mounting tool H. 8. Check that membrane D is pressed flat (not folded) against the opening in the electrode holder A by inspecting it against a light source.

  • Page 7: Tpp + Membrane Conditioning And Storage

    MiPNet15.03 O2k-MultiSensor-ISE 6. The bulb should protrude noticeable from the electrode tip. The size of the bulb can be controlled by screwing part C more or less into part B. A good result is usually obtained by fully Bulb ok inserting part C into part B.

  • Page 8: Wash The Ise

    A very effective cleaning procedure is immersion of the electrode in a solution of living or dead cells (surplus from cell cultures) or tissue homogenates in the O2k- chamber. If necessary, this should be performed after rinsing with (1) water, (2) ethanol, and (3) water.

  • Page 9: Reference Electrode: Assembly, Storage And Maintenance

    MiPNet15.03 O2k-MultiSensor-ISE insufficiently respond to the analyte (even after re- conditioning in the storage solution) the membrane must be exchanged. 2.5. Reference electrode: assembly, storage and maintenance The Reference-Electrode\2.4 mm for ISE is composed of an internal silver-silver chloride electrode with an internal filling solution of 3 M KCl saturated with AgCl.

  • Page 10: O2K-Multisensor System

    3. O2k-MultiSensor system The O2k-FluoRespirometer supports all add-on O2k- Modules and includes all O2k-MultiSensor channels mentioned below. For O2k- Series B and C see Appendix. Before handling the BNC plugs (on the O2k-Main Unit) and connecting the electrodes, always touch the...

  • Page 11: Operating Instructions

    ESD. Connect: Insert the plug of the ISE into the BNC plug labelled “pX” on the front of the O2k-Main Unit, and the plug of the reference electrode into the 2 mm pin plug labelled “Ref” (MiPNet19.18A). Gain:...

  • Page 12: Volume Calibration With Ise-Multisensor Stoppers

    4.2. Volume calibration with ISE-MultiSensor stoppers When using an ISE-MultiSensor stopper, the ISE and reference electrodes must be in place when calibrating the O2k-chamber volume, comparable to volume- calibration with standard stoppers (MiPNet19.18A). Mitochondria and cell research Oroboros Instruments...

  • Page 13: Experiment

    MiPNet15.03 O2k-MultiSensor-ISE Add to the dry O2k-Chamber containing the stirrer bar a water volume accounting for the final chamber volume (2 mL) plus the additional dead volume in the capillary and spaces between electrodes and inlets. For the Oroboros ISE Assembly (ion selective electrode and...

  • Page 14: Instrumental Background Oxygen Flux

    MultiSensor experiments. 4.4.1. Dithionite background Because of difficulties involved in opening and closing the O2k-Chamber with an ISE-MultiSensor Stopper, it is strongly recommended to use the instrumental background procedure based on dithionite injections (MiPNet14.06) to avoid repeated opening and closing of the O2k-Chamber.

  • Page 15: Ise-Calibration And Performance Test

    100 mM KCl solution. The calibration runs should be performed right before a biological experiment using experimental medium. In the case of a TPP electrode being used to determine membrane potential, ideally the biological sample is injected into the O2k-chamber directly after TPP calibration. typical ISE-calibration...
  • Page 16 0.1 mM solution is used. 4.5.4. calibration with the TIP2k Fill the O2k-chambers with medium and close the chamber with electrodes inserted as described above. Fill the TIP syringes with the calibration solution and insert the TIP needles into the chambers.

  • Page 17: Performance Criteria

    MiPNet15.03 O2k-MultiSensor-ISE TIP2k titrations: Start calibration titrations with the TIP2k after a stable signal is obtained. The following TIP2k setup can be applied, starting at 0.7 µM TPP Why do you not simply give the name of the TiP2k setup...

  • Page 18: Membrane Lifetime

    A membrane should only be replaced when performance longer satisfactory. O2k-MultiSensor control and calibration 5.1. pX signal Graphs can be constructed to include both, recorded oxygen and pX, or several graphs can be added to display oxygen and pX data separately. All graph settings can be saved as user-defined layouts (MiPNet19.18C).

  • Page 19: Configuration And Gain

    In the O2k configuration window the pX electrode is entered for documentation. The gain for the pX channel is set in the O2k control window [F7], tab Potentiometric, pX to 10, 20, 40, or 80. The gain amplifies the “pX Raw Signal”.

  • Page 20: Calibration

    MiPNet15.03 O2k-MultiSensor-ISE 5.3. Calibration In the example of TPP calibration with TiP2k, the values of -6.155, -6.046, -5.959, -5.886, -5.824 correspond to LogTPP concentrations 0.7, 0.9, 1.1, 1.3, 1,5∙10 M respectively. The concentrations were used as mark names and Log of these concentrations for concentration in the marks specifications.
  • Page 21 MiPNet15.03 O2k-MultiSensor-ISE original calibration (e.g. pH) was performed initially or a file in which these values were applied. Then calibrate with Calibrate and copy to clipboard. Reset to raw signal: It is often desirable to set the calibrated signal equal to the raw signal. This can be done any...

  • Page 22: Supplement A: Datlab 7.1

    MiPNet15.03 O2k-MultiSensor-ISE Supplement A: DatLab 7.1. O2k configuration window: pX calibration window: High-resolution respirometry Oroboros Instruments...
  • Page 23 MiPNet15.03 O2k-MultiSensor-ISE The graph shows linear dependence of raw signal on Log[TPP ], the slope represents the sensitivity of the TPP electrode in [V/1]. The real sensitivity is calculated by dividing the sensitivity [V/1] by gain. In the example the sensitivity is 1.1907/20 = 0.05953 V per unit of Log[TPP ] or 59.53...

  • Page 24: Determine The O2K Series

    O2k series. The series is specified as the first character of the serial number of the O2k, printed on the sticker on the rear of the O2k housing (MiPNet19.18A). A serial number B-#### or C-#### denotes an Oxygraph from the B or C series, while D-#### denotes an Oxygraph from the D series and so on.

  • Page 25: O2K Series B And C, Px Upgrade Installed Before 2011

    MiPNet15.03 O2k-MultiSensor-ISE O2k series B and C, pX upgrade installed before 2011 Connect: Two electrodes (measuring + reference electrode) are connected to the MultiSensor BNC plug of the O2k (Series B and C) through a MultiSensor Connector. The MultiSensor Connector has a black cable with a...

  • Page 26: O2K Series B And C, Px Upgrade Installed After 2010

    O2k series B and C, pX upgrade installed after 2010 Connect: For O2k series B to C with a pX upgrade installed after 2010, reference electrodes directly...

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