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Difference between revisions of "O2k-TPP+ ISE-Module"

From Bioblast
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== '''O2k-TPP+ and Ca2+ ISE-Module ''consists of'':''' ==
== '''O2k-TPP<sub>+</sub> ISE-Module ''consists of'':''' ==
{{#ask: mainlabel=Title|[[Category:Products]] | [[Product type::O2k-TPP-Ca ISE-Module]]  
{{#ask: mainlabel=Title|[[Category:Products]] | [[Product type::O2k-TPP-Ca ISE-Module]]  
| ?description
| ?description
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== O2k-Core and O2k Series A-E ==
== O2k-Core and O2k Series A-E ==


For the Oxygraph-2k Series A-E, the '''O2k-TPP<sup>+</sup> and Ca<sup>2+</sup> ISE-Module''' is supported by the [[O2k-Core]] and by all O2k instruments with the [[O2k-MultiSensor|O2k-MultiSensor electronic upgrade]].
For the Oxygraph-2k Series A-E, the '''O2k-TPP<sup>+</sup> ISE-Module''' is supported by the [[O2k-Core]] and by all O2k instruments with the [[O2k-MultiSensor|O2k-MultiSensor electronic upgrade]].


= TPP /TPMP electrode =
= TPP /TPMP electrode =


The main purpose of the O2k-TPP+ and Ca2+ ISE-Module is to measure  [[Mitochondrial membrane potential]] via the TPP or TPMP method.
The main purpose of the O2k-TPP+ ISE-Module is to measure  [[Mitochondrial membrane potential]] via the TPP or TPMP method.


The first publication using the O2k-TPP+ and Ca2+ ISE-Module for this purpose was [[Brown 2011 Am J Physiol Regul Integr Comp Physiol]]
The first publication using the O2k-TPP<sup>+</sup> ISE-Module for this purpose was [[Brown 2011 Am J Physiol Regul Integr Comp Physiol]]


== Calibration of the TPP electrode ==
== Calibration of the TPP electrode ==
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* If you have a "Multimeter" available set in into "Voltmeter mode"  
* If you have a "Multimeter" available set in into "Voltmeter mode"  
* Set the recording range of the voltmeter to a range good for recording about 200 mV
* Set the recording range of the voltmeter to a range good for recording about 200 mV
* now measure the voltage difference between reference electrode and TPP electrode by touching (ideally fixing) the two probes of       the voltmeter to the ends of the cables from the electrodes: for the reference electrodes this is simply the gold colored pin, for       the TPP electrode this is the INNER , central pin. Note down the voltage reading on the voltmeter display. For this step the help      of a second person is most convenient, otherwise you probable have to find  a way to fix the cables in some way. It is not necessary to have the voltmeter in contact with the electrodes all the time, just when you are reading the values.
* now measure the voltage difference between reference electrode and TPP electrode by touching (ideally fixing) the two probes of the voltmeter to the ends of the cables from the electrodes: for the reference electrodes this is simply the gold colored pin, for the TPP electrode this is the INNER , central pin. Note down the voltage reading on the voltmeter display. For this step the help      of a second person is most convenient, otherwise you probable have to find  a way to fix the cables in some way. It is not necessary to have the voltmeter in contact with the electrodes all the time, just when you are reading the values.
* Increase the TPP concentration in a few (lets say 5) rather large steps (at least 2 µM per step) and note down the displayed      values.
* Increase the TPP concentration in a few (lets say 5) rather large steps (at least 2 µM per step) and note down the displayed      values.
* Finally reconnect the electrodes to the pX electronics of the O2k and observe how much (if at all) the signal has changed there.      * Please send us the recorded values along with the TPP concentrations used and the the DatLab file recorded at the same time (that shows the readings for the first and last point). If no change in signal is visible with the voltmeter, the problem is at the TPP electrode, not the O2k.
* Finally reconnect the electrodes to the pX electronics of the O2k and observe how much (if at all) the signal has changed there.      * Please send us the recorded values along with the TPP concentrations used and the the DatLab file recorded at the same time (that shows the readings for the first and last point). If no change in signal is visible with the voltmeter, the problem is at the TPP electrode, not the O2k.

Revision as of 10:31, 11 November 2014

                



O2k-TPP+ ISE-Module

O2k-Catalogue

Description O2k-TPP+ ISE-Module: 2 potentiometric ion-selective electrodes (ISE), ISE-Service Kit and 2 PVDF-Stoppers, supported by the O2k-Core.
Product ID 12300-01
Type O2k, O2k-Module, MultiSensor, Stopper, Catalogue
Link O2k-Multisensor @OROBOROS, ISE-Manual, Tetraphenylphosphonium
Image
O2k-TPP-ISE.jpg




O2k-TPP+ ISE-Module consists of:

The query description has an empty condition.

General

The OROBOROS ISE system is designed with replaceable membranes making it possible to measure different ions such as TPP+ or Ca2+ with the same electrode housing.

O2k-Core and O2k Series A-E

For the Oxygraph-2k Series A-E, the O2k-TPP+ ISE-Module is supported by the O2k-Core and by all O2k instruments with the O2k-MultiSensor electronic upgrade.

TPP /TPMP electrode

The main purpose of the O2k-TPP+ ISE-Module is to measure Mitochondrial membrane potential via the TPP or TPMP method.

The first publication using the O2k-TPP+ ISE-Module for this purpose was Brown 2011 Am J Physiol Regul Integr Comp Physiol

Calibration of the TPP electrode

Data export and linear calibration

Mark stable sections on the pX raw signal, use or create a template of mark names, and copy to clipboard in Marks Statistics [F2]. Copy into an Excel template for TPP+ calibration. This template can be modified according to the specific calibration experiment (titration volumes, concentrations, number of data points, …). Perform a linear regression of the pX raw signal as a function of the log10 (analyte concentration). For highest accuracy, only the concentration range used in the final experiment should be included in the regression. The Excel template provides information on the regression parameter, R2, and by the deviation of data points from the regression (the residuals), and can be adjusted to specific calibration regimes.



From the O2k-MultiSensor menu the MultiSensor calibration window [Ctrl+F5] is opened MiPNet12.08 for a simple two-point linear calibration of pX (or –pX) as a function of recorded voltage, using known pX values.


Calcium electrode

We recommend to use fluorescence methods for measuring Ca2+ concentrations, utilizing the O2k-Fluorescence LED2-Module. Fluorescence based methods for detection of Ca2+ are more easy to set up and are more widely used in biosciences than ISE based methods. However, there may be special applications in which determination of Ca2+ levels via ISE is advantageous. The use of a Ca2+ electrode in mitochondrial research was described by Moreno et al. [1] Also a laboratory already using the O2k-TPP+_and_Ca2+_ISE-Module but without funds for the O2k-Fluorescence LED2-Module may want to use ISE based Ca2+ detection. For this purposes ISE-Ca2+ Membranes suitable for use with the O2k-TPP+_and_Ca2+_ISE-Module can be ordered from OROBOROS INSTRUMENTS and are described here:

A Ca2+ selective membrane with a composition similar to the one described by Simon and coworkers [2] has been tested with the OROBOROS ISE system and detection limits of less than 10 nM were achieved in Ca2+ buffered calibration solutions. There may be further optimizations of the membrane composition to ideally fit the the typical applications of OROBOROS customers. However, the main obstacle in establishing useful Ca2+ measurements appears to be not the Ca2+ sensitive membrane but the methodology of its application in a medium simulating intracellular conditions.

Note: The operation of a Ca2+ electrode at typically high extracellular Ca2+ concentrations is straightforward and devoid of the difficulties described here. These difficulties include both the calibration of the Ca2+ electrode and the planning of a biological experiment to use it.

ISE-Ca2+ Membranes for use in the Oroboros ISE system can be ordered separately.

Operation of the Ca2+ electrode

General

The general operation (assembly, ...) of the Oroboros ISE system is described in MiPNet15.03.

While working with the pX channel please always observe the guidelines for avoiding damage to the electronics by ESD.

Calculation of free Ca2+ concentrations

To work at physiological Ca2+ concentrations requires usually Ca2+ buffering by chelating agents. The calculation of c(Ca2+)free in such solutions is quite complicated and the results depend i.a on ionic strength, temperature, and - very strongly - on the pH. Tools for such calculations are available on-line at Chris Pattons MAXCHELATOR Page maxchelator.stanford.edu. Many calculations, published or implemented in programs, depend on a single set of data compiled by Martell and Smith [3]. Note that in spite of the availability of easy to use software tools the calculation of free Ca2+ concentration is not simple. The accuracy of these calculations is severely limited by several factors:

  • Many underlying thermodynamic constants may not be known precisely
  • Some thermodynamic constants necessary for adequate temperature correction may not be known at all
  • It may be difficult to set the experimental parameters pH and ionic strength with the necessary precision.

Some strategies to simplify the necessary calculations plus a method for the preparation of Ca buffers are presented in Tsien R, Pozzan T (1989) Measurement of cytosolic free Ca2+ with quin2. Methods Enzymol 172: }, 230-262. The Calcium calibration buffer kit (C3008MP) available from Life Technologies (former Invitrogen) is based on this publication.


Inner filling solution

The following inner filling solution is used: CaCl2: 10 mM

EDTA: 50 mM

pH adjusted to 8.5 with KOH


Conditioning

Conditioning of the membrane is a controversial topic. If any conditioning is done, the used free Ca2+ concentration should probable not be much higher than the highest expected concentration during applications of the electrode.

Calibration

As a potentiometric method, the Ca2+ electrode delivers a signal that is (in the working range) linear to the logarithm of the free Ca2+ concentration. Therefore, the electrode is calibrated by plotting electrode signal vs. logarithm of the free Ca2+ concentration. Calibration of the Ca2+ electrode at low (< 1µM) Ca2+ levels is typically done by exposing the electrode to a series of Ca2+ calibration buffers. Each calibration solution typically contains a Ca2+ chelating agent, a pH buffer, CaCl2, and a salt (KCl) to adjust ionic strength. For suggested compositions see the literature in the reading list below. Examples from the literature may then be adapted to individual needs using the software tools mentioned above. The pH of all solutions have to be adjusted very carefully. For calibrations spanning a large range (several orders of magnitude) of free Ca2+ concentrations it is necessary to use calibration buffers with very different compositions. The nonlinearity of the electrode response in such calibrations may be mainly due to the difficulties in calculating the free Ca2+ concentration.

Application in biological experiments

You could help us in the development by explaining what you would like to do/ see in a Ca2+ experiment and what you expect from the electrode: what ca concentrations do you want to measure, Ca2+ release or uptake? what total concentration change in a 2 ml chamber (!) do you expect? Do you want to measure in a Ca buffered medium? (decreased sensitivity to changes) or without Ca2+ buffering but then how do you get to physiological Ca2+ concentration? Do you want to measure at physiological Ca2+ concentrations?.......

  • Please add your comments in the Discussion page. Pease contact instruments@oroboros.at to recive an account.

Reading list

We have complied a short reading list that may be of interest to those planing to to do Ca2+ measurements, with special emphasis on references describing the preparation of Ca2+ calibration buffers.

Ca measurement reading list


Advanced Diagnostic tests for the ISE system

In case of problems please follow first the suggestions in the chapter "Trouble shooting" of the manual. Below are some further options that may help if the procedures described in the manual were already tried. Sometimes it is difficult to find out whether problems with the ISE system are caused by the electrodes (TPP, Ca, reference, pH) or by the pX electronics of the O2k. The following tests can help to solve this question.

Testing a TPP system outside the O2k-Chamber

This test allows to test for any disturbing influences in the chamber, like crosstalk to the OroboPOS. It is therefore similar to the test proposed in the manual (switching off the O2 polarization voltages) but has a broader scope (covering also e.g. possible interference from stirrers) and provides a more complete separation of POS an pX electrodes.

Place TPP electrode and reference electrode together in one falcon tube filled with electrolyte solution (e.g. media). Connect the cables of the electrodes to the O2k and record the pX signal. If a problem previously observed disappears in this test (and is reproducible by using the same electrodes in the O2k-Chamber) the problem is located in the chamber, e.g. at a leaky POS membrane, see the manual.


Testing a TPP system with a pH electrode (and vice versa)

This method allows to test the sensitivity of the pX electronics.

Requirements

Another type of electrode than can be connected to the ISE system. So if the problem is observed with an ISE system for measuring TPP, a pH electrode is the usual choice because it is available in many labs. The only requirements for the second electrode is that it can be connected via a BNC port. It does not have to be an electrode from OROBOROS Instruments neither is it necessary that the electrode fits into the O2k chamber. However, note that using a pH electrode outside the O2k-Chamber also constitutes a test similar to the one described above (Testing a TPP system outside the O2k-Chamber). Therefore a problem caused e.g. by a leaky POS membrane will not be observed when testing with a pH electrode outside the chamber.

Procedure

  • Connect the pH electrode to the oxygraph.
  • Put the electrode into a pH 4 calibration buffer (event)
  • Observe (record) the signal in DatLab.
  • Put the electrode into a pH 7 calibration buffer (event). Obviously the sequence of buffers does not matter.
  • From such a DatLab file we will see whether the pX electronics is working and even get a very rough estimation of the gain.

Testing TPP or pH electrodes with a voltmeter

This test measures the performance of the electrodes independently form the pX electronics of the O2k.

Requirements

Any voltmeter, frequently labeled "Multimeter" if different measurement modes can be selected, suitable for measuring mV potentials. .

Procedure for TPP electrodes

  • Set up the TPP /reference electrode in the O2k chamber as usually, only medium in the chamber.
  • Connect the electrodes to the pX electronics and record the signal.
  • Disconnect both electrode cables from the O2k (but leave the electrodes in the chamber, stirring still on).
  • If you have a "Multimeter" available set in into "Voltmeter mode"
  • Set the recording range of the voltmeter to a range good for recording about 200 mV
  • now measure the voltage difference between reference electrode and TPP electrode by touching (ideally fixing) the two probes of the voltmeter to the ends of the cables from the electrodes: for the reference electrodes this is simply the gold colored pin, for the TPP electrode this is the INNER , central pin. Note down the voltage reading on the voltmeter display. For this step the help of a second person is most convenient, otherwise you probable have to find a way to fix the cables in some way. It is not necessary to have the voltmeter in contact with the electrodes all the time, just when you are reading the values.
  • Increase the TPP concentration in a few (lets say 5) rather large steps (at least 2 µM per step) and note down the displayed values.
  • Finally reconnect the electrodes to the pX electronics of the O2k and observe how much (if at all) the signal has changed there. * Please send us the recorded values along with the TPP concentrations used and the the DatLab file recorded at the same time (that shows the readings for the first and last point). If no change in signal is visible with the voltmeter, the problem is at the TPP electrode, not the O2k.

Procedure for pH electrodes

  • Follow the procedure for TPP electrodes, moving the pH electrode between different pH calibration buffers instead of doing a TPP titration.

References

  1. Moreno AJM, Vicente JA (2012) Use of a calcium-sensitive electrode for studies on mitochondrial calcium transport. Methods Mol Biol 810: 207-217.
  2. Schefer U, Ammann D, Pretsch E, Oesch U, Simon W (1986) Neutral carrier based Ca2+-selective electrode with detection limit in the sub-nanomolar range. Analyt Chem 58: 2282-2285.
  3. Martell AE, Smith RM (1989) Critical Stability Constants, 1: Amino Acids. Plenum Press




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