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| '''''Under construction'''''
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| = Calcium and HRR =
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| == Options for measuring Ca<sup>2+</sup> ==
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| We discuss here measuring Ca<sup>2+</sup> concentrations via fluorometry (using [[Calcium green]] fluorophores) or via an ion selective electrode (ISE) system. We recommend to use fluorescence methods for measuringĀ Ca2+ concentrations. Fluorescence based methods are more easy to set up and areĀ more widely used in biosciences than ISE based methods.Ā However, thereĀ may be special applications for whichĀ determination ofĀ Ca2+Ā levels via ISE is advantageous. The use of a Ca2+ electrode in mitochondrial research was described by Moreno et al.Ā <ref>Moreno AJM, Vicente JA (2012) Use of a calcium-sensitiveĀ electrode for studies on mitochondrial calcium transport. Methods Mol Biol 810:207-17.</ref>Ā
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| On this page we discuss topics related to the measurement of Ca2+ that apply to all available methods.
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| :Ā» [[Calcium green]]: fluorophores from the Calcium green family for measurement of Ca<sup>2+</sup> concentrations with the [[O2k-Fluorometer]].
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| :Ā» [[O2k-TPP%2B_ISE-Module#Calcium_electrode]]: Measurement of Ca<sup>2+</sup> with the OROBOROS ISE module.
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| == How to start a Ca2+ project ==
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| Please have a look at [[O2k-Publications: Calcium]].
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| Another good starting pointĀ (and not only for a fluorescence based approach but also for measurements based on ISE) are the web pages of Invitrogen (see main page forĀ 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,Ā Ca2+ calibration buffers) are discussed in more detail below. Points to be addressedĀ will be:
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| * Can the problem be tackled by using one of the fluorophores from Invitrogene's [[Calcium green]] series? Which of them?
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| * What kind of methods are published?
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| WhatĀ the [[O2k-Fluorescence LED2-Module]] actually does, is to bring the functionality ofĀ a cuvette based spectrofluorometer (for selected excitation and emissionĀ wavelengths) to the O2k chamber. Therefore a publication using a cuvette basedĀ measurement of a Calcium green fluorophore should also be doable in theĀ O2k with the fluorescence module. In contrast, the fluorescence moduleĀ is not intended to supply the functions of e.g. a fluorescenceĀ microscope (with which e.g. it is possible to quantify the fluorescenceĀ 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 theĀ 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 changes inĀ fluorophore intensity generated inside intact cells (or mitochondria ?) might be measured. This will require the the use of cell permeable fluorophores and application of the cell loading techniques described by the fluorophores producer, see also [[Talk:Calcium_green#Cell_permeable_Ca2.2B_fluorophores]].
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| IdeallyĀ initial experiments will follow published methods but with theĀ additional dimensions of simultaneous measurement of respiration /Ā control of oxygen concentration. A publication that can be used as aĀ starting point should:
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| * address the problems you are interested in,
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| * use one of the differentĀ [[Calcium green]] fluorophores,
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| * use a cuvette based approach in a (spectro)fluorometer (or, of course, use the [[O2k-Fluorescence LED2-Module]], see [[Elustondo 2014 Biochim Biophys Acta]]).
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| With more experience it may be possible to follow publications that use aĀ different Ca2+ fluorophore by substituting it with a suitable [[Calcium green]]Ā derivative.
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| We haveĀ complied a short reading list that may be ofĀ interest to those planing to do Ca<sup>2+</sup>Ā measurements, with special emphasisĀ on references describing theĀ preparation of Ca<sup>2+</sup>Ā calibration buffers.
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| [[Media:Ca_reading_list.pdf|Ca measurement reading list]]
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| == Ca2+ buffering ==
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| MitochondriaĀ are easily damaged even by the low calcium levels resulting fromĀ impurities in chemicals and preparations. In fact the nativeĀ intracellular Ca2+ level may be below the total Ca2+ concentration in aĀ medium introduced as impurities of standard laboratory chemicals. InĀ respirometric experiments this is taken care of by "buffering away" allĀ the Ca2+ with e.g. EGTA. But buffering and measuring small differencesĀ are of course at cross purpose. One strategy is first usingĀ a very weak Ca2+ buffering, just to keep Ca2+ impurities under controlĀ and then add external Ca2+ in sufficient amounts to "out-titrate" theĀ buffer. In the subsequent part of the experiment the mitochondria are exposed to un-physiologically high Ca2+ concentrations anyway.
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| == Calculation of free Ca<sup>2+</sup> concentrations ==
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| ToĀ work at physiological Ca<sup>2+</sup> concentrationsĀ requires usually Ca<sup>2+</sup> buffering by chelatingĀ agents. The calculation ofĀ ''c''(Ca<sup>2+</sup>)<sub>free</sub> in suchĀ solutions is quite complicated and the results depend i.a on ionicĀ strength, temperature, and - very strongly - on the pH. Tools for suchĀ calculations are available on-line at Chris Pattons MAXCHELATOR PageĀ [http://maxchelator.stanford.edu/Ā maxchelator.stanford.edu] or theĀ [http://web.stanford.edu/~cpatton/webmaxcS.htmĀ web version]. ManyĀ calculations, published or implemented in programs, depend on a singleĀ set of data compiled by Martell and Smith <ref>Martell AE, SmithĀ RM (1989) Critical Stability Constants, 1: Amino Acids. PlenumĀ Press</ref>.
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| Note that in spite of the availability of easyĀ to use software tools the calculation of freeĀ Ca<sup>2+</sup> concentration is not simple. The accuracy ofĀ these calculations is severely limited by several factors:
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| * Many underlying thermodynamic constants may not be known precisely.
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| * Some thermodynamic constants necessary for adequate temperature correction may not be known at all.
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| * It may be difficult to set the experimental parameters pH and ionic strength with the necessary precision.
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| SomeĀ strategies to simplify the necessary calculationsĀ are presented in <ref name="Tsien 1989"> Tsien R, Pozzan T (1989)Ā Measurement of cytosolic free Ca2+ with quin2. Methods Enzymol 172:230-62. </ref>.
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| == Ca2+ calibration ==
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| Before deciding on a calibration method (or specific buffers from thisĀ method) one should carefully access the needs of the project underĀ consideration. Is an absolute quantification of Ca2+ necessary at all? Which Ca2+ concentrationsĀ should beĀ measured? Maybe one wants to measure extracellular Ca2+ levels? - thenĀ noĀ Ca2+ buffered calibration solution will be necessaryĀ at all. Is it possible to do a Ca2+ calibration in the medium used forĀ the experiment, ie. is it possible to perform the experiment in medium veryĀ similar to the Ca2+ calibration solutions or can the Ca2+ calibration solutions be modified to be very similar to the used medium?
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| CalibrationĀ at low (<Ā 1ĀµM)Ā Ca<sup>2+</sup> levels isĀ typically done by using a series of Ca<sup>2+</sup>Ā calibrationĀ buffers. Each calibration solution typically contains a Ca<sup>2+</sup> chelating agent, a pH buffer,Ā Ā CaCl<sub>2</sub>, and a salt (KCl) to adjust ionic strength. The pH of all solutionsĀ have to be adjusted very carefully.Ā ForĀ calibrations spanning a largeĀ range (several orders of magnitude) ofĀ free Ca<sup>2+</sup>Ā concentrations it may be necessary toĀ use calibration buffers with veryĀ different compositions. UnexpectedĀ features in calibration curve may in this case mainly due to theĀ difficulties in calculatingĀ the free Ca<sup>2+</sup>Ā concentration, leading to different errors for different mediaĀ compositions.
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| A comparable simple method for theĀ preparation of Ca2+ buffers is presented by Tsien et al. <ref name="Tsien 1989" />. The Calcium calibration buffer kit (C3008MP)Ā 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]].Ā
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| == Application in biological experiments ==
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| Please help us in our instrumental development by explaining what you would like toĀ do/ see in a Ca<sup>2+</sup> experiment and what you expectĀ from the measuring method: What Ca concentrations do you want to measure,Ā Ca<sup>2+</sup> release or uptake? What total concentrationĀ change in a 2 ml chamber (!) do you expect? Do you want to measure in aĀ Ca buffered medium? (decreased sensitivity to changes) or withoutĀ Ca<sup>2+</sup> buffering but then how do you get toĀ physiological Ca<sup>2+</sup> concentration? Do you want toĀ measure at physiological Ca<sup>2+</sup>Ā concentrations?.......
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| Please add your comments inĀ the [[Calcium|Discussion page]]. Please contactĀ instruments@oroboros.at to set up an account.
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| == References ==
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| <references/>
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| <br>
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| {{#set:Technical service=Ca}}
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| [[Category:Technical service]]
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| [[Category:All]]
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