Calcium

high-resolution terminology - matching measurements at high-resolution

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

MitoPedia methods: Fluorometry 

under construction !

Ca2+ and HRR

Options for measuring Ca2+

We discuss here measuring Ca2+ concentrations using fluorescence or an ion selective electrode (ISE) system. We recommend to use fluorescence methods for measuring Ca2+ concentrations. 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. On this page we discuss topics related to the measurement of Ca2+ that apply to all available methods. Please see Calcium green for specific information about using the fluorophore Ca green to measure Ca2+ concentrations with the O2k-Fluorescence LED2-Module. Please see O2k-TPP+_ISE-Module#Calcium_electrode for specific information about measuring Ca2+ with an ISE system.

How to start a Ca2+ project

Please have a look at O2k-Publications: Calcium.

Another good starting point (and not only for a fluorescence based approach but also for measurements based on ISE) are the web pages of Invitrogen (see main page for links)and publications cited there. Reading existing calcium related publications should give you a feeling for the issues involved. Some of them (the necessity of Ca2+ buffering, Ca2+ calibration buffers) are discussed in more detail below. Points to be addressed will be:

• Can the problem be tackled by using one of the fluorophores from Invitrogene's Calcium green series? Which of them?
• What kind of methods are published?

What the O2k-Fluorescence LED2-Module actually does, is to bring the functionality of a cuvette based spectrofluormeter (for selected excitation emission wavelengths) to the O2k chamber. So a publication using a cuvette based measurement of a Calcium green fluorophore should also be doable in the O2k with the fluorescence module. In contrast, the fluorescence module is not intended to supply the functions of e.g. a fluorescence microscope (with which e.g. it is possible to quantify the fluorescence from a single cell A cuvette based approach (and therefore also one using the O2k-Fluorescence LED2-Module) will always observe the entire medium in the cuvette / chamber, most of it will be OUTSIDE of the mitos/ cells. Therefore, frequently concentrations outside of the sample are monitored and a fluorophore is chosen that does not permeate cell membranes. However, if the background fluorescence from outside the mitochondria is small enough (or constant enough) also change in fluorophore intensity generated inside intact cells (or mitochondria ?) might be measured. This will require the the use of cell permeable fluorophores and the cell loading techniques described by the fluorophores producers.

Ideally initial experiments will follow published methods but with the additional dimensions of simultaneous measurement of respiration / control of oxygen concentration. A publication that can be used as a starting point should:

• address the problems you are interested in,
• use one of the different Calcium green fluorophores,
• use a cuvette based approach in a (spectro)fluorometer.

With a bit more experience it may be possible to follow publications that use a different Ca2+ fluorophore by substituting it with a suitable Calcium green derivative.

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

Ca2+ buffering

Mitochondria are easily damaged even by the low calcium levels resulting from impurities in chemicals and preparations. In fact the native intracellular Ca2+ level may be below the total Ca2+ concentration in a medium introduced as impurities of standard laboratory chemicals. In respirometric experiments this is taken care of by "buffering away" all the Ca2+ with e.g. EGTA. But buffering and measuring small differences are of course at cross purpose. One strategy is first using a very weak Ca2+ buffering, just to keep Ca2+ impurities under control and then add external Ca2+ in sufficient amounts to "out-titrate" the buffer. In the subsequent part of the experiment the mitochondria are exposed to un-physiologically high Ca2+ concentrations anyway.

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 or the web version. Many calculations, published or implemented in programs, depend on a single set of data compiled by Martell and Smith . 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 are presented in .

Ca2+ calibration

Before deciding on a calibration method (or specific buffers from this method) one should carefully access the needs of the project under consideration. Is an absolute quantification of Ca2+ necessary at all? Which Ca2+ concentrations should be measured? Maybe one wants to measure extracellular Ca2+ levels? - then no Ca2+ buffered buffered calibration solution will be necessary at all. Is it possible to do a Ca2+ calibration in the medium used for the experiment, ie. is it possible to perform the experiment in medium very similar to the Ca2+ calibration solutions or can the Ca2+ calibration solutions be modified to be very similar to the used medium?

Calibration at low (< 1µM) Ca²⁺ levels is typically done by using 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. 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 may be necessary to use calibration buffers with very different compositions. Unexpected features in calibration curve may in this case mainly due to the difficulties in calculating the free Ca²⁺ concentration, leading to different errors for different media compositions.

A comparable simple method for the preparation of Ca2+ buffers is presented by Tsien et al. . The Calcium calibration buffer kit (C3008MP) available from Life Technologies (former Invitrogen) is based on this publication. The manual for this calcium calibration kit can be read as a general introduction in performing Ca2+ calibrations following Tsiens approach, even if the required solutions are prepared in the laboratory and not purchased. Other recipes for Ca2+ calibration buffers can be found in the literature cited in the Ca measurement reading list.

Application in biological experiments

Please help us in our instrumental development by explaining what you would like to do/ see in a Ca²⁺ experiment and what you expect from the measuring method: 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. Please contact [email protected] to set up an account.