Fluorescent Mg²⁺ Indicators—Section 19.6

Page Contents

• Magnesium Indicators Excited by UV Light
• Magnesium Indicators Excited by Visible Light
• Data Table
• Ordering Information

Intracellular Mg²⁺ is important for mediating enzymatic reactions, DNA synthesis, hormonal secretion and muscular contraction. To facilitate the investigation of magnesium's role in these and other cellular functions, we offer several different fluorescent indicators for measuring intracellular Mg²⁺ concentration. They include furaptra, which we refer to as mag-fura-2 to denote the similarity of its structure and spectral response with the Ca²⁺ indicator fura-2; and mag-indo-1, with a structure and spectral response similar to that of indo-1. For applications such as confocal laser-scanning microscopy and flow cytometry, we offer the Magnesium Green and mag-fluo-4 indicators. The various methods for measuring intracellular Mg²⁺ have been reviewed.

Mg²⁺ indicators are generally designed to maximally respond to the Mg²⁺ concentrations commonly found in cells, typically ranging from about 0.1 mM to 6 mM. Intracellular free Mg²⁺ levels have been reported to be ~0.3 mM in synaptosomes, 0.37 mM in hepatocytes and 0.5–1.2 mM in cardiac cells, whereas the concentration of Mg²⁺ in normal serum is ~0.44–1.5 mM. Measurements using fluorescent Mg²⁺ indicators are somewhat more demanding than intracellular Ca²⁺ determinations because physiological changes in Mg²⁺ concentration are relatively small. Compartmentalization and binding to proteins can also be a problem in use of these indicators in cells. Mg²⁺ indicators also bind Ca²⁺; however, typical physiological Ca²⁺ concentrations (10 nM–1 µM) usually do not interfere with Mg²⁺ measurements because the affinity of these indicators for Ca²⁺ is low. For ultrasensitive Mg²⁺ measurement applications, intracellular Ca2+ background can be suppressed using BAPTA AM (B1205, B6769; B1205, B6769; Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). AlthoughCa²⁺ binding by Mg²⁺ indicators can be a complicating factor in Mg²⁺ measurements, this property can also be exploited for measuring high Ca²⁺ concentrations (1–100 µM); see Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2 and Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3 for further examples.

For intracellular calibration of Mg²⁺ indicators, we offer the ionophore A-23187 (A1493, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), which are preferred over ionomycin (I24222) because they transport Mg²⁺ more effectively. Solutions used to calibrate Mg²⁺ indicators should be initially free of heavy metals such as Mn²⁺ that can interact with the indicators. These metals can be removed by treating the solution with the divalent cation chelator TPEN (Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8).

Mag-Fura-2 and Mag-Indo-1

The dissociation constant for Mg²⁺ of mag-indo-1 is 2.7 mM, slightly higher than that of mag-fura-2, which is 1.9 mM. The lower-affinity mag-indo-1 indicator is sensitive to somewhat higher spikes in intracellular Mg²⁺. The affinities of mag-fura-2 and mag-indo-1 for Mg²⁺ are reported to be essentially invariant at pH values between 5.5 and 7.4 and at temperatures between 22°C and 37°C. A detailed study of the photophysics of mag-fura-2 has been published. Comparisons of intracellular and solution dissociation constants for mag-fura-2 have been published by Günther and by Tashiro and Konishi.

As with their Ca²⁺ indicator analogs, mag-fura-2 undergoes an appreciable shift in excitation wavelength upon Mg²⁺ binding (Figure 19.6.1), and mag-indo-1 exhibits a shift in both its excitation and emission wavelengths (Figure 19.6.2). Equipment, optical filters and calibration methods are very similar to those required for the Ca²⁺ indicators. The excitation-ratioable mag-fura-2 indicator is most useful for fluorescence microscopy, whereas the emission-ratioable mag-indo-1 indicator is preferred for flow cytometry. Many applications of mag-fura-2 involve estimation of the affinity and selectivity of Mg²⁺ binding to proteins. Displacement of bound Mg²⁺ by Li⁺ provides a surrogate assay for Li⁺ transport, a process for which few direct detection methods exist. Researchers have used mag-fura-2 to measure intracellular Mg²⁺ in a wide variety of cells, organelles and tissues, including:

• Cortical neurons
• Isolated mitochondria
• Platelets
• Rat hepatocytes
• Rat ventricular myocytes
• Xenopus oocytes

In addition to the cell-impermeant potassium salt of mag-fura-2 (M1290), we offer the cell-permeant AM ester of mag-fura-2 as a set of 20 vials, each containing 50 µg (M1292). The special packaging is recommended when small quantities of the dyes are to be used over a long period of time.

We also offer visible light–excitable Mg²⁺ indicators, including the Magnesium Green and mag-fluo-4 indicators. As with mag-fura-2 and mag-indo-1, these visible light–excitable Mg²⁺ indicators can also be used as low-affinity Ca²⁺ indicators (Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) and may be useful as indicators for Zn²⁺ and other metals (Fluorescent Indicators for Zn2+ and Other Metal Ions—Section 19.7).

Magnesium Green Indicator

The Magnesium Green indicator exhibits a higher affinity for Mg²⁺ (K_d ~1.0 mM) than does mag-fura-2 (K_d ~1.9 mM) or mag-indo-1 (K_d ~2.7 mM); this indicator also binds Ca²⁺ with moderate affinity (K_d for Ca²⁺ in the absence of Mg²⁺ is ~6 µM, measured at 22°C using our Calcium Calibration Buffer Kits). The spectral properties of the Magnesium Green indicator are similar to those of the Calcium Green indicators. Upon binding Mg²⁺, Magnesium Green exhibits an increase in fluorescence emission intensity without a shift in wavelength (Figure 19.6.3). The Magnesium Green indicator has been used to investigate the binding of free Mg²⁺ by the bacterial SecA protein and by protein tyrosine kinases. By exploiting the fact that ATP has greater Mg²⁺-binding affinity than ADP, researchers have used Magnesium Green to indirectly measure ATP in pancreatic acinar cells, in bullfrog hair cells, in cultured Xenopus spinal neurons and in isolated mitochondria. Magnesium Green is available as a cell-impermeant potassium salt (M3733) or as a cell-permeant AM ester (M3735).

Mag-Fluo-4

Mag-fluo-4 is an analog of fluo-4 with a K_d for Mg²⁺ of 4.7 mM and a K_d for Ca²⁺ of 22 µM (measured at 22°C using our Calcium Calibration Buffer Kits), making it useful as an intracellular Mg²⁺ indicator as well as a low-affinity Ca²⁺ indicator (Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3). Mag-fluo-4 has a much more sensitive fluorescence response to Mg²⁺ binding than does our Magnesium Green indicator. Because physiological fluctuations of intracellular Mg²⁺ concentration are typically small, this increased sensitivity is a considerable advantage. Like fluo-4, mag-fluo-4 is essentially nonfluorescent in the absence of divalent cations and exhibits strong fluorescence enhancement with no spectral shift upon binding Mg²⁺ (Figure 19.6.4). Mag-fluo-4 is available as a cell-permeant AM ester (M14206).

For a detailed explanation of column headings, see Definitions of Data Table Contents