Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ macroelectrodes

McGuigan, John A. S.; Stumpff, Friederike

doi:10.1016/j.ab.2012.12.020

Cited by 7 publications

(3 citation statements)

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“…The manufacture of buffers by the ratio method, although straightforward, requires care and technical skill. If a series of buffers is manufactured and the [Ca 2+ ]/[Mg 2+ ] in each of the 10 buffers is measured, then the coefficient of variation [CV = (SD/mean) × 100] can be taken as a measure of the accuracy of manufacture (McGuigan & Stumpff, : table 2). During the development of the LOM, the CV for EGTA buffers was 27.4% (McGuigan et al., ), later reduced to ≤7% for EGTA and BAPTA buffers owing to an increase in the accuracy of weighing, pipetting, pH a control and in the manufacture of the solutions (McGuigan & Stumpff, ).…”

Section: Measurement Of [Ca2+]/[mg2+] In Buffer Solutionsmentioning

confidence: 99%

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Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated

McGuigan

Kay

Elder

2020

Experimental Physiology

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New Findings What is the topic of this review?The [Ca2+]/[Mg2+] in buffers are usually calculated using one of eight programs. These all give different values, thus [Ca2+]/[Mg2+] must be measured. What advances does it highlight?The ligand optimization method (LOM) using electrodes is an accurate method to do this. The limitations of the method are described. The LOM has been generalized to include calibration of fluorochromes and aequorin. It is the method of choice to measure intracellular equilibrium constants. Owing to the uncertainties for the values of resting [Ca2+], ∆[Ca2+] and the pK′ values for intracellular Ca2+/Mg2+ binding used in modelling, these values must now be re‐examined critically. Abstract Modelling intracellular regulation of Ca2+/Mg2+ is now an established part of physiology. However, the conclusions drawn from such studies depend on accurate knowledge of intracellular [Ca2+], ∆[Ca2+] and the pK′ values for the intracellular binding of Ca2+/Mg2+. Calculation of [Ca2+]/[Mg2+] in buffers is normal. The eight freely available programs all give different values for the [Ca2+]/[Mg2+] in the buffer solutions, varying by up to a factor of 4.3. As a result, concentrations must be measured. There are two methods to do this, both based on the ligand optimization method (LOM): (1) calibration solutions from 0.5 to 4 mmol l−1; and (2) calibration solutions from 0.1 µmol l−1 to 2 mmol l−1. Both methods can be used to calibrate Ca2+/Mg2+ electrodes. Only Method 2 can be used directly to calibrate fluorochromes and aequorin. Software in the statistical program R to calculate the [Ca2+]/[Mg2+] in buffers is provided for both methods. The LOM has now been generalized for use with electrodes, fluorochromes and aequorin, making it the ideal method to determine the pK′ values for intracellular binding of Ca2+/Mg2+. The [Ca2+]/[Mg2+] in buffers must be measured routinely, which is best done by calibrating electrodes with the LOM and software written in R. If [Ca2+]/[Mg2+] in buffers are calculated, the parameters used in modelling show the same degree of variability as the software programs. Uncritical acceptance of such parameters means that conclusions reached from such studies are relative, not absolute, and must now be re‐examined.

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Section: Measurement Of [Ca2+]/[mg2+] In Buffer Solutionsmentioning

confidence: 99%

“…The plot is for a Ca 2+ ion-selective electrode (ISE) fromMcGuigan and Stumpff (2013). The slope, s, was 28.533 mV per decade and Σ 6.045 × 10 −8 mol l −1 .…”

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confidence: 99%

Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated

McGuigan

Kay

Elder

2020

Experimental Physiology

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“…Despite well‐established thermodynamic models, predicted free metal ion concentrations can be associated with significant uncertainty, introduced mainly by the choice of constants, ambiguities concerning their correction and the lack of international standards or clearly defined protocols for metal ion buffer preparation. This is exemplified by experimental and modelling studies on traditional Ca 2+ and Mg 2+ buffers, which identified several sources of uncertainty and potential error. They comprise (i) the use of empirically derived thermodynamic constants and the frequent lack of enthalpy values for proper temperature correction, (ii) the use of non‐thermodynamic conventions to calculate single ion activity coefficients for ionic strength correction, (iii) errors in the conversion between measured pH (ie H + activity) and proton concentration due to liquid junction potential changes at the reference pH electrode and (iv) uncertainties with regard to ligand purity, pipetting errors and the level of contaminating metal ions present.…”

Section: Theory Of Trace Metal Ion Bufferingmentioning

confidence: 99%

A practical guide to the preparation and use of metal ion‐buffered systems for physiological research

et al. 2017

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Recent recognition that mobile pools of Zn and Cu are involved in the regulation of neuronal, endocrine and other cells has stimulated the development of tools to visualize and quantify the level of free trace metal ions. Most of the methods used to measure or control loosely bound metals require reference media that contain exactly defined free concentrations of the target ions. Despite the central importance of proper metal ion buffering, there is still a lack of international standards and beginners in the field may have difficulties finding a coherent description of how to prepare trace metal ion buffers, especially when experiments are to be performed in multimetal systems. To close this gap, we provide a guide for the design, preparation and use of metal ion-buffered systems that facilitate immediate application under physiologically relevant ionic conditions. Thermodynamic and kinetic concepts of chemical speciation as well as general protocols and specific examples are outlined for the accurate preparation of single- and dual-metal ion buffers. In addition, experiments have been performed with FluoZin-3 to illustrate that metal ion-buffered systems are required for reliable preparation of nanomolar Zn solutions and that dual-metal ion buffers can be used to calibrate suitable fluorescent Zn sensors in the presence of millimolar Ca concentrations. Together, the information provided should sensitize readers to the many potential pitfalls and uncertainties that exist when working with physiologically relevant concentrations of trace metal ions and enable them to formulate their own metal ion buffers for most in vitro applications.

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Simultaneous Membrane Capacitance Measurements and TIRF Microscopy to Study Granule Trafficking at Immune Synapses

2017

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Whole-cell capacitance measurements allow the direct measurement of exocytosis with high temporal resolution. An added benefit of the whole-cell configuration is the possibility to control the cytosolic free calcium concentration allowing examination of the role of intracellular calcium in a variety of processes. We have coupled this method with imaging of cytotoxic granule release using total internal reflection fluorescence microscopy (TIRFM) to identify the capacitance steps associated with cytotoxic granule release identified by TIRFM. This requires the use of fluorescent granule markers to identify cytotoxic granules and allows characterization of cytotoxic granule fusion and of the behavior of cytotoxic granules at the immune synapse prior to fusion. Combination of these methods enables the study of a number of processes relevant to the function of the immune synapse.

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Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ macroelectrodes

Cited by 7 publications

References 11 publications

Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated

Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated

A practical guide to the preparation and use of metal ion‐buffered systems for physiological research

Simultaneous Membrane Capacitance Measurements and TIRF Microscopy to Study Granule Trafficking at Immune Synapses

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Calculated and measured [Ca2+] in buffers used to calibrate Ca2+ macroelectrodes

Cited by 7 publications

References 11 publications

Ionized concentrations in Ca2+ and Mg2+ buffers must be measured, not calculated

Ionized concentrations in Ca2+ and Mg2+ buffers must be measured, not calculated

A practical guide to the preparation and use of metal ion‐buffered systems for physiological research

Simultaneous Membrane Capacitance Measurements and TIRF Microscopy to Study Granule Trafficking at Immune Synapses

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Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated

Ionized concentrations in Ca²⁺ and Mg²⁺ buffers must be measured, not calculated