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 ionophores A-23187 and the nonfluorescent 4-bromo A-23187 (A1493, B1494; 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 (T1210, 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:
In addition to the cell-impermeant potassium salt of mag-fura-2 (M1290), we offer the cell-permeant AM esters of mag-fura-2 and mag-indo-1 as a set of 20 vials, each containing 50 µg (M1292, M1295). The special packaging is recommended when small quantities of the dyes are to be used over a long period of time. Mag-fura-2 AM is also available in a single vial containing 1 mg (M1291).
Figure 19.6.1 A) Fluorescence excitation and B) fluorescence emission spectra of mag-fura-2 (M1290) in solutions containing 0–35 mM Mg2+.
Figure 19.6.2 A) Fluorescence excitation and B) fluorescence emission spectra of mag-indo-1 in solutions containing 0–100 mM Mg2+.
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+ (Kd ~1.0 mM) than does mag-fura-2 (Kd ~1.9 mM) or mag-indo-1 (Kd ~2.7 mM); this indicator also binds Ca2+ with moderate affinity (Kd 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).
Figure 19.6.3 Mg2+-dependent fluorescence emission spectra of Magnesium Green (M3733).
Mag-fluo-4 () is an analog of fluo-4 with a Kd for Mg2+ of 4.7 mM and a Kd 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-impermeant potassium salt (M14205) or as a cell-permeant AM ester (M14206).
Figure 19.6.4 Fluorescence emission spectra of mag-fluo-4 (M14205) in solutions containing 0–50 mM Mg2+.
|Low Mg2+||High Mg2+|
|M1290||586.68||F,D,L||pH >6||369||22,000||511||H2O||330||24,000||491||H2O/Mg2+||1.9 mM||1, 2, 3, 4|
|mag-indo-1||594.74||F,D,L||pH >6||349||38,000||480||H2O||330||33,000||417||H2O/Mg2+||2.7 mM||1, 2, 3, 4|
|M3733||915.90||F,D,L||pH >6||506||77,000||531||H2O||506||75,000||531||H2O/Mg2+||1.0 mM||1, 2, 3, 4, 5|
|M14205||681.77||F,D,L||pH >6||490||74,000||see Notes||H2O||493||75,000||517||H2O/Mg2+||4.7 mM||1, 2, 3, 4, 6|