Calcium

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MitoPedia

Calcium

Description

Ca2+ 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.

Abbreviation: Ca

Reference: O2k-Publications: Calcium

Oroboros support

O2k-technical support

This information is part of O2k-technical support and open innovation.

Fluorometric measurement of Ca

» Calcium Green


Potentiometric measurement with ion selective electrode (ISE)

Ca measurement

O2k-TPP+ ISE-Module: ISE-Ca2+ Membranes are not included in the O2k-TPP+ ISE-Module but can be ordered from Oroboros Instruments separately (Product ID 42280-01), for application of the O2k-TPP+ ISE-Module for Ca2+ measurements.
The Oroboros ion selective electrode (ISE) is designed with replaceable membranes making it possible to measure different ions such as Ca2+, TPP+, TPMP+ with the same electrode housing. See also Calcium for general consideration about Ca2+ measurements. We recommend to use fluorescence methods for measuring Ca2+ concentrations, utilizing the O2k-Fluo_LED2-Module. 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] Laboratories who are using the O2k-TPP+ ISE-Module (but who do not have the O2k-Fluo_LED2-Module) may apply the ISE for Ca2+ detection.

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. For a discussion of Ca2+ calibration buffers see Tsien & Pozzan.
Calculation of free Ca2+ concentrations:» [1] or [2]

Ca2+ electrode and FCCP

There are strong indications that FCCP and the ISE-Ca2+ Membranes are incompatible.


References

  1. Moreno AJM, Vicente JA (2012) Use of a calcium-sensitive electrode for studies on mitochondrial calcium transport. Methods Mol Biol 810:207-17.



MitoPedia methods: Fluorometry