Calcium: Difference between revisions

Latest revision as of 15:03, 20 April 2021

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 of the 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 O2k and high-resolution respirometry: O2k-Open Support

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 Smart-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 Smart-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]

Ca²⁺ electrode and FCCP

Some experimental evidence indicates that FCCP and the ISE-Ca2+ Membranes are incompatible.

References

↑ 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

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

[1]

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=Ca
-|description='''Ca<sup>2+</sup>''' 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 Ca<sup>2+</sup> levels  is thus of considerable interest in the context of [[High-resolution respirometry]].
+|description='''Ca<sup>2+</sup>''' 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 of the entire cell. Measuring changes in Ca<sup>2+</sup> levels  is thus of considerable interest in the context of [[high-resolution respirometry]].
 |info=[[O2k-Publications: Calcium]]
 }}
-{{MitoPedia methods
-|mitopedia method=Fluorometry
-}}
-{{MitoPedia topics}}
 __TOC__
+{{Template:Technical support integrated}}
+== Fluorometric measurement of Ca ==
+::::» [[Calcium Green]]
-<big>'''under construction !'''</big>
-= 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.  <ref>Moreno AJM, Vicente JA (2012) Use of a calcium-sensitive  electrode for studies on mitochondrial calcium transport. Methods Mol  Biol 810: 207-217.</ref>
-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%2B_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 Ca<sup>2+</sup>  measurements, with special emphasis  on references describing the  preparation of Ca<sup>2+</sup>  calibration buffers.
-[[Media:Ca_reading_list.pdf|Ca measurement reading list]]
+== 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<sup>+</sup> ISE-Module for Ca<sup>2+</sup> measurements.
-== Ca2+ buffering ==
+:::: The Oroboros ion selective electrode (ISE) is designed with replaceable membranes making it possible to measure different ions such as Ca<sup>2+</sup>, TPP<sup>+</sup>, TPMP<sup>+</sup> with the same electrode housing. See also [[Calcium]] for general consideration about Ca<sup>2+</sup> measurements. We recommend to use fluorescence methods for measuring Ca<sup>2+</sup> concentrations, utilizing the [[O2k-Fluo Smart-Module]]. However, there may be special applications in which determination of Ca<sup>2+</sup> levels via ISE is advantageous. The use of a Ca<sup>2+</sup> electrode in mitochondrial research was described by Moreno et al.<ref>Moreno AJM, Vicente JA (2012) Use of a calcium-sensitive electrode for studies on mitochondrial calcium transport. Methods Mol Biol 810:207-17.</ref> Laboratories who are using the [[O2k-TPP+ ISE-Module| O2k-TPP<sup>+</sup> ISE-Module]] (but who do not have the [[O2k-Fluo Smart-Module]]) may apply the ISE for Ca<sup>2+</sup> detection.
-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.
+=== Inner filling solution ===
+:::: The following inner filling solution is used:
+::::* CaCl<sub>2</sub>: 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 Ca<sup>2+</sup> concentration should probable not be much higher than the highest expected concentration during applications of the electrode.
-== Calculation of free Ca<sup>2+</sup> concentrations ==
+=== Calibration ===
-To  work at physiological Ca<sup>2+</sup> concentrations  requires usually Ca<sup>2+</sup> buffering by chelating  agents. The calculation of  ''c''(Ca<sup>2+</sup>)<sub>free</sub> 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  [http://maxchelator.stanford.edu/  maxchelator.stanford.edu] or the   [http://web.stanford.edu/~cpatton/webmaxcS.htm  web version]. Many  calculations, published or implemented in programs, depend on a single  set of data compiled by Martell and Smith <ref>Martell AE, Smith  RM (1989) Critical Stability Constants, 1: Amino Acids. Plenum  Press</ref>.
+:::: As a potentiometric method, the Ca<sup>2+</sup> electrode delivers a signal that is (in the working range) linear to the logarithm of the free Ca<sup>2+</sup> concentration. Therefore, the electrode is calibrated by plotting electrode signal vs. logarithm of the free Ca<sup>2+</sup> concentration. Calibration of the Ca<sup>2+</sup> electrode at low (< 1µM) Ca<sup>2+</sup> levels is typically done by exposing the electrode to a series of Ca<sup>2+</sup> calibration buffers. For a discussion of Ca<sup>2+</sup> calibration buffers see [https://www.ncbi.nlm.nih.gov/pubmed/2747529 Tsien & Pozzan].
-Note that in spite of the availability of easy  to use software tools the calculation of free  Ca<sup>2+</sup> concentration is not simple. The accuracy of  these calculations is severely limited by several factors:
+:::: Calculation of free Ca<sup>2+</sup> concentrations:» [https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/downloads.htm]
-* 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 <ref name="Tsien 1989"> Tsien R, Pozzan T (1989)  Measurement of cytosolic free Ca2+ with quin2. Methods Enzymol 172: 230-262. </ref>.
-== Ca2+ calibration ==
+=== Ca<sup>2+</sup> electrode and FCCP ===
-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?
+:::: Some experimental evidence indicates that FCCP and the [[ISE-Ca2+ Membranes]] are incompatible.
-Calibration  at low (<  1µM)  Ca<sup>2+</sup> levels is  typically done by using a series of Ca<sup>2+</sup>  calibration   buffers. Each calibration solution typically contains a    Ca<sup>2+</sup> chelating agent, a pH buffer,    CaCl<sub>2</sub>, 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 Ca<sup>2+</sup>   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 Ca<sup>2+</sup>  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. <ref name="Tsien 1989" />. The Calcium calibration buffer kit (C3008MP)  available from Life Technologies (former Invitrogen) is based on this publication. The [https://tools.lifetechnologies.com/content/sfs/manuals/CalciumCalibrationBufferKits_PI.pdf 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 [[Media:Ca_reading_list.pdf|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 Ca<sup>2+</sup> experiment and what you expect  from the measuring method: What Ca concentrations do you want to measure,  Ca<sup>2+</sup> 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  Ca<sup>2+</sup> buffering but then how do you get to  physiological Ca<sup>2+</sup> concentration? Do you want to  measure at physiological Ca<sup>2+</sup>  concentrations?.......
-Please add your comments in  the [[Calcium|Discussion page]]. Please contact  [email protected] to set up an account.
 == References ==
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 <references/>
-<br>
+{{MitoPedia methods
-{{#set:Technical service=Ca}}
+|mitopedia method=Fluorometry
+}}
-[[Category:Technical service]]
-[[Category:All]]