Fluorescent Indicators for Zn²⁺ and Other Metal Ions—Section 19.7

Page Contents

• Applications of Ca2+ and Mg2+ Indicators for Detection of Zn2+ and Other Metals
• Indicators for Zinc
• Indicators for Copper
• Indicators for Iron
• Indicators for Lead, Cadmium and Mercury
• Indicators for Nickel and Cobalt
• Indicators for Aluminum
• Indicators for Lanthanides
• Data Table
• Ordering Information

Not only do transition metal ions play an important role in biological structure and activity, but they can also serve as surrogates and blockers of ion transport through Ca²⁺ channels (Figure 19.7.3). Measuring heavy metal ion concentrations in cells and environmental samples with indicators originally designed for detection of Ca²⁺ and Mg²⁺ has been hampered by competitive binding of other, more abundant, cations. Detection methods rely both on novel fluorescent ion sensors specifically designed for metal detection, as well as on new applications for indicators originally designed for detection of Ca²⁺ and Mg²⁺ (Response of fura-2 and indo-1 to some divalent cations other than Ca2+ and Mg2+—Table 19.5, Figure 19.7.1, Figure 19.7.2).

Several of our fluorescent indicators can be used to selectively determine polyvalent cation concentrations inside cells or to follow metal ion transport through ion channels. Other indicators are primarily useful for measurements in solutions or in extracts of environmental samples. In most cases, the high affinity of the indicators for metal ions allows interference from other compounds to be minimized by diluting the sample with deionized water. As with Ca²⁺ and Mg²⁺ detection, spectroscopic responses to metal ion binding and dissociation constant (K_d) values are dependent on many factors, including pH, temperature, viscosity, protein binding and the presence of other ions. These responses may vary significantly in complex environments such as seawater and the cytosol.

We have tested the responses of many of the Ca²⁺ and Mg²⁺ indicators (described in Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2 through Fluorescent Mg2+ Indicators—Section 19.6) to a series of polyvalent metal ions (Figure 19.7.1, Figure 19.7.2). The BAPTA-based indicators, including the Calcium Green dyes, fluo-3 and fluo-4, exhibit the highest emission intensities upon binding La³⁺, Hg²⁺ and Cd²⁺. The response of the Calcium Green-1 and Magnesium Green indicators shows relatively little variability amongst the ions tested. Indicators nominally designed for detecting low Ca²⁺ concentrations, such as Calcium Green-5N, fluo-5N and rhod-5N, show little response to transition metal ions such as Fe²⁺, Co²⁺, Ni²⁺ and Co²⁺ and much stronger responses to heavier ions such as Cd²⁺, Hg²⁺ and Pb²⁺. Note that the responses shown in Figure 19.7.2 are not necessarily saturated or even uniform in some cases. Some indication of the overall response pattern can be obtained by comparing the effects of the 1 µM and 100 µM ion concentrations sampled in these experiments. Also, note that our selection of excitation and emission wavelengths for the measurements may significantly affect the absolute and relative magnitudes of the changes observed.

Fura-2 and indo-1, like other BAPTA-based Ca²⁺ indicators, are highly selective for Ca²⁺ over Mg²⁺; however, they bind other divalent and trivalent cations with significantly higher affinity. In terms of spectroscopic detection, some of these ions (e.g., Ba²⁺, Cd²⁺ and Sr²⁺) mimic the effects of Ca²⁺; others (e.g., Mn²⁺, Co²⁺ and Ni²⁺) exert strong fluorescence quenching effects (Response of fura-2 and indo-1 to some divalent cations other than Ca2+ and Mg2+—Table 19.5). Although heavy metal cations are a potentially serious source of interference in Ca²⁺ measurements using fluorescent indicators, intracellular concentrations are fortunately very low in most cases. When interference occurs, it can be identified and controlled using the selective heavy metal ion chelator TPEN. Quenching of indicator fluorescence by Mn²⁺ has several useful applications:

• Calibrating the intracellular Ca²⁺ response of fluo-3, fluo-4, rhod-2 and related indicators
• Discriminating fluorescence signals from mitochondrial and cytosolic indicator populations
• Estimating the fraction of residual Ca²⁺-insensitive indicators after AM ester loading
• Estimating indicator leakage in measurements on cell suspensions in cuvettes

Mn²⁺ is also extensively used as an ionic surrogate for measuring Ca²⁺ influx through ion channels (Figure 19.7.3). Because the effect of Mn²⁺ on fura-2 fluorescence is quite different from that of Ca²⁺ (Response of fura-2 and indo-1 to some divalent cations other than Ca2+ and Mg2+—Table 19.5), influx can be clearly distinguished from Ca²⁺ elevation due to mobilization of intracellular stores, although such clarity may be impaired by the existence of alternative Mn²⁺-influx pathways. A similar analysis applies when using fura-2 and indo-1 to study the antagonistic effects of ions such as Ni²⁺, La³⁺ and Co²⁺ on voltage-activated and Ca²⁺ release–activated Ca²⁺ channels. In this case, the Ca²⁺-dependent fluorescence signal is of primary interest and permeability of the antagonist ion, resulting in direct interactions with the indicator, will usually invalidate the measurement. For the Calcium Green, Oregon Green 488 BAPTA and Magnesium Green indicator series, the response to Mn²⁺ is not markedly different from the Ca²⁺ response (Figure 19.7.2), making these indicators generally unsuitable for the applications based on Ca²⁺/Mn²⁺ discrimination described above. A variety of fluorescent indicators, including fura-2, fura-FF and our Calcium Orange and Magnesium Green indicators, have been utilized to detect Sr²⁺, an ion that can replace Ca²⁺ in triggering neurotransmitter release and also serves as a blocker of mitochondrial permeability transition pore opening.

Fluorescence of calcein (C481) is quenched strongly by Co²⁺, Ni²⁺ and Cu²⁺ and appreciably by Fe³⁺ and Mn²⁺ at physiological pH (panel A, Figure 19.7.4). This fluorescence quenching response can be exploited for detecting the opening of the mitochondrial permeability transition pore, for assaying membrane fusion (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3) and for monitoring cellular iron transport and the cellular labile iron pool.

Zinc is the second most abundant transition metal in living organisms after iron. It is of particular importance in the regulation of gene expression, as Zn²⁺ binding proteins account for nearly 50% of the transcription regulatory proteins in the human genome. Zn²⁺ is also functionally active in pancreatic insulin secretion and is a contributory factor in neurological disorders including epilepsy and Alzheimer disease. Free Zn²⁺ is released from metalloprotein complexes during oxidative stress. The intracellular concentration of free Zn²⁺ is extremely low in most cells (<1 nM), with the remainder being bound to proteins or nucleic acids. One calculation yields an intracellular free Zn²⁺ concentration of six orders of magnitude less than one atom per cell. We prepare a variety of Zn²⁺ indicators (Fluorescent indicators for Zn2+—Table 19.6) to help elucidate the role of Zn²⁺ release and the localization of free or chelatable Zn²⁺ in cells.

FluoZin Indicators

Zinc concentrations in the 1–100 nM range can be measured using fluorescent indicators nominally designed for Ca²⁺ detection such as fura-2 (see below), or more recently developed indicators with greater Zn²⁺ selectivity. We have focused our development efforts on probes for detection of higher Zn²⁺ concentrations that are present in synaptic vesicles and released in response to electrical stimulation or excitotoxic agonists. Peak concentrations of synaptically released Zn²⁺ may exceed 100 µM. We have developed FluoZin-1 and FluoZin-2 (F24189), a series of unique indicators designed for detection of Zn²⁺ in the 0.1–100 µM range with minimal interfering Ca²⁺ sensitivity (panel H, Figure 19.7.2; Figure 19.7.5). In our laboratories, we determined a K_d(Zn²⁺) of 8.2 µM for FluoZin-1; however, dissociation constants are known to vary considerably depending on the experimental conditions and a K_d(Zn²⁺) of 0.4 µM for this same indicator has been reported elsewhere, underscoring the importance of calibrating the indicator directly in the system under study. Published applications of FluoZin-1 have primarily involved characterization of Zn²⁺-binding proteins.

The FluoZin-3 indicator (F24194, ; F24195) is a Zn²⁺-selective indicator with a structure that resembles fluo-4. FluoZin-3 exhibits high Zn²⁺-binding affinity (K_d for Zn²⁺ ~15 nM) that is unperturbed by Ca²⁺ concentrations up to at least 1 µM. The Zn²⁺-specificity of FluoZin-3 evident from measurements in calibration solutions is reproduced in cell-based experiments. The responses of FluoZin-3 to transition metals, including Zn, Mn, Fe, Co, Cu(I), Cu(II), Ni and Cd, have been extensively characterized. In addition, FluoZin-3 exhibits a large increase in fluorescence in response to saturating levels of Zn²⁺ (greater than 50 fold, Figure 19.7.6). FluoZin-3 has been found to be a brighter alternative to Zinquin for measuring the exocytotic release of Zn²⁺ from pancreatic β-cells after stimulation with glucose or potassium. Although imaging applications of FluoZin-3 are predominant, cell-based microplate assays and flow cytometry protocols have also been developed.

RhodZin-3 Indicator

The orange-fluorescent RhodZin-3 zinc indicator exhibits a dramatic 75-fold increase in fluorescence at saturating levels of Zn²⁺ and also possesses a K_d for Zn²⁺ of ~65 nM. The cell-permeant AM ester form of RhodZin-3 effectively localizes into mitochondria and is a valuable tool for investigating the physiological consequences of mitochondrial Zn²⁺ sequestration. The cell-impermeant RhodZin-3 salt has been used to measure extracellular Zn²⁺ concentrations.

Newport Green DCF and Newport Green PDX Indicators

The Newport Green DCF indicator (N7991) has moderate zinc-binding affinity (K_d for Zn²⁺ ~1 µM) but is essentially insensitive to Ca²⁺ (K_d for Ca²⁺ >100 µM), making this a valuable probe for detecting Zn²⁺ influx into neurons through voltage- or glutamate-gated channels. When used alongside dyes with dual Ca²⁺/Zn²⁺ sensitivity such as fura-2 and mag-fura-2, Newport Green DCF provides confirmation that changes in Zn²⁺ levels, and not Ca²⁺ or Mg²⁺, are being detected. Newport Green DCF has been used to identify insulin-producing β-cells from human pancreatic islets based on their high intracellular Zn²⁺ content (). Newport Green DCF has also been used in conjunction with Texas Red 10,000 MW dextran to create a ratiometric fluorescent PEBBLE (Probe Encapsulated By Biologically Localized Embedding) nanosensor for real-time measurements of intra- and intercellular free zinc. The response of Newport Green DCF to transition metals, including Zn, Mn, Fe, Co, Cu(I), Cu(II), Ni and Cd, have been characterized.

Newport Green PDX incorporates the same di-(2-picolyl)amine chelator as Newport Green DCF but has a higher Zn²⁺ dissociation constant (K_d for Zn²⁺ ~30 µM) and a larger Zn²⁺-free to Zn²⁺-saturated fluorescence intensity increase.

TSQ

Use of the membrane-permeant probe N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) in cells was first described by Fredrickson. TSQ is selective for Zn²⁺ in the presence of physiological concentrations of Ca²⁺ and Mg²⁺ ions. The complex of TSQ with free Zn²⁺ apparently has a stoichiometry of two dye molecules per metal atom, but a 1:1 complex may be formed with metalloproteins. The intracellular Zn²⁺ chelator dithizone blocks TSQ binding of Zn²⁺.

Several reports suggest that TSQ can be used to localize Zn²⁺ pools in the central nervous system. Histochemical localization using TSQ identified a broad distribution of Zn²⁺ in neonatal mice, particularly associated with rapidly proliferating tissues, such as skin and gastrointestinal epithelium. TSQ has also been used to detect Zn²⁺ translocation from presynaptic nerve terminals into postsynaptic nerve terminals when blood flow is constricted in the brain during ischemic events. TSQ (like Newport Green DCF, see above) is a selective nontoxic stain for pancreatic islet cells, which have a high content of Zn²⁺, and may be useful for their flow cytometric isolation.

TSQ-based assays for Zn²⁺ in seawater and other biological systems exhibit a detection limit of ~0.1 nM. The simultaneous determination of Zn²⁺ and Cd²⁺ by spectrofluorometry using TSQ in an SDS micelle has also been reported. TSQ has been used to measure Zn²⁺ levels in artificial lipid vesicles and live sperm cells by flow cytometry. In this latter study, the fluorescence yield of the TSQ–Zn²⁺ complex was shown to be much higher when bound to lipids than in aqueous solution, indicating that quantitative cell assays for Zn²⁺ based on the fluorescence intensity of TSQ may not be accurate because of uncertainty in the quantum yield of the dye when bound to membranes.

Traditional Ca²⁺ and Mg²⁺ Indicators as Zn²⁺ Indicators

Zn²⁺ binds to most BAPTA-based Ca²⁺ indicators with substantially higher affinity than Ca²⁺. For example, fura-2 (F1200, F6799; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2) exhibits a K_d for Zn²⁺ in the absence of Ca²⁺ of 3 nM (Response of fura-2 and indo-1 to some divalent cations other than Ca2+ and Mg2+—Table 19.5). The lack of saturation of fura-2 fluorescence in resting cells is indicative of the low intracellular concentration of free Zn²⁺. Fura-2 remains sensitive to nanomolar Zn²⁺ levels in the presence of 25–100 nM free Ca²⁺, allowing the use of fura-2 AM (F1201, F1221, F1225, F14185; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2) for detecting intracellular Zn²⁺ influx via voltage-gated Ca²⁺ channels and for examining Zn²⁺ levels in live neurons and myocytes. Fura-2 has also been used as an indicator for nanomolar levels of free Zn²⁺ and other ions in solution.

Mag-fura-2 (M1290, Fluorescent Mg2+ Indicators—Section 19.6) exhibits slightly altered spectral characteristics upon binding Zn²⁺ (K_d ~20 nM at pH 7.0–7.8 and 37°C), allowing Zn²⁺ to be measured in the presence of Ca²⁺. A review by Dineley and co-workers evaluates the performance of currently available fluorescent indicators for neuronal Zn²⁺ and identifies artifacts associated with their use. As usual, the key to indicator selection is matching the ion binding affinity (K_d) to the prevailing range of ion concentrations. In the case of neuronal free Zn²⁺, physiological concentrations apparently are about 1–50 nM. Under these conditions, indicators nominally designed for detection of intracellular magnesium, such as mag-fura-2 with K_d for Zn²⁺ around 20 nM, appear to be the most suitable. Based on similar ion-binding properties, the Magnesium Green indicator (M3733, Fluorescent Mg2+ Indicators—Section 19.6) should be useful for confocal imaging of Zn²⁺ influx.

Copper is third in abundance (after Fe³⁺ and Zn²⁺) among the essential heavy metals in the human body. Dietary copper is required for normal hemoglobin synthesis, for prevention of anemia and for redox enzyme activity. The redox activity of copper (i.e., reversible reduction of Cu²⁺ to Cu⁺) is both a key to its biological activity and a complicating factor in its detection. Reduction of Cu²⁺ to Cu⁺ is catalyzed by the β-amyloid precursor protein, which is converted to plaques that are characteristic of Alzheimer disease.

Phen Green FL for Cu²⁺

The phenanthroline-based Phen Green FL indicator, available only as the cell-permeant diacetate (P6763), is an excellent general-purpose heavy metal sensor capable of detecting a broad range of metal ions, including both Cu²⁺ and Cu⁺. The use of Phen Green FL for detecting Fe²⁺, Hg²⁺, Pb²⁺, Cd²⁺ and Ni²⁺ at submicromolar concentrations is described below. Uncomplexed Phen Green FL is brightly fluorescent, with a fluorescence quantum yield of ~0.8. Binding of certain heavy metal ions is registered by strong fluorescence quenching (Figure 19.7.4). The emission intensity of Phen Green FL depends both on metal ion concentration and on the indicator's concentration. Phen Green FL diacetate has allowed researchers to discern significant differences in intracellular Cu²⁺ levels for four types of lobster hepatopancreatic epithelial cells.

Phen Green SK for Cu⁺

The phenanthroline-based Phen Green SK indicator (P14312) has been reported to be a selective indicator for Cu⁺, using conditions that minimize the oxidation of Cu⁺ to Cu²⁺. These researchers then used Phen Green SK to characterize the dissociation of Cu⁺ from glutathione.

Traditional Ca²⁺ and Mg²⁺ Indicators as Cu²⁺ Indicators

Fluorescence of calcein (C481) is strongly quenched by Cu²⁺ at neutral pH (Figure 19.7.4), although this spectroscopic response does not appear to have been exploited for Cu²⁺ detection. Cu²⁺ has also been measured in solution using fura-2. Indicators designed for detection of Ca²⁺, Mg²⁺ and Zn²⁺ generally exhibit relatively weak responses to Cu²⁺. FluoZin-3 and Newport Green DCF bind Cu²⁺ with extremely high affinity (K_d = 0.09 nM and 0.8 nM, respectively). Cu²⁺ binding is registered as a fluorescence decrease due to competitive displacement of Zn²⁺. For both indicators, binding affinity for Cu⁺ is more than 1000-fold weaker than for Cu²⁺.

Phen Green FL and Phen Green SK Indicators

The intracellular pool of chelatable iron is considered to be a decisive pathogenic factor for various kinds of cell injury. Fluorescence of Phen Green FL (available only as the cell-permeant diacetate, P6763) and Phen Green SK (P14312, ; cell-permeant diacetate, P14313) is quenched upon binding Fe²⁺ (Figure 19.7.1) and Fe³⁺ (Figure 19.7.4). The emission intensity of the Phen Green FL indicator depends on both the metal ion's concentration and the indicator's concentration. Phen Green SK diacetate has been successfully used to quantitate the intracellular pool of chelatable iron in rat hepatocytes, to directly measure Fe²⁺ transport across chloroplast membranes and to monitor iron uptake in relation to fatigue in mouse skeletal muscle.

Calcein

Cabantchik and co-workers have exploited the fluorescence quenching of calcein at neutral pH to follow intracellular release of Fe²⁺ from transferrin. Using passively loaded calcein AM (C1430, C3099, C3100MP), they were able to measure cytosolic Fe²⁺ concentrations from about 0.1 µM to 1.0 µM. However, more recent studies on the mechanism of iron chelation indicate that quenching of calcein fluorescence is primarily due to Fe³⁺ and is relatively insensitive to Fe²⁺ (Figure 19.7.4). Nontransferrin-bound iron (NTBI) occurs in the serum of individuals with iron overload and in a variety of other pathological conditions, including thalassemia. A microplate assay based on the fluorescence quenching of calcein by iron has been developed to measure NTBI. The capacity of transition metals such as iron to catalyze the generation of oxidative radicals can be used as a basis for indirect detection using fluorescent ROS sensors (Generating and Detecting Reactive Oxygen Species—Section 18.2) under conditions where the metal ion is the limiting species. Dihydrorhodamine 123 (D632, D23806; Generating and Detecting Reactive Oxygen Species—Section 18.2) has been used in this way to detect NTBI in the plasma of healthy and thalassemic patients.

Leadmium Green Indicator

Leadmium Green dye is a fluorescent indicator supplied in cell-permeant AM ester form (A10024) for selectively detecting lead or cadmium in cells. The calcium-insensitive Leadmium Green dye can detect nanomolar levels of lead and micromolar levels of cadmium (Figure 19.7.7). In a typical application, Leadmium Green AM was used for assessment of the effects of N-acetylcysteine on intracellular lead levels in oligodendrocyte progenitor cells.

Measure-iT Lead and Cadmium Assay Kit

The Measure-iT Lead and Cadmium Assay Kit (contact Custom Services for more information) provides a fluorescence microplate assay for quantitation of lead or cadmium in solution, with a linear detection range from 5 to 200 nM. (Figure 19.7.8). The Measure-iT Leadmium reagent working solution is mixed with 1–20 µL sample volumes in the wells of a microplate and then immediately analyzed with a fluorescence microplate reader using fluorescein/FITC wavelength settings. The assay is performed at room temperature, and the signal is stable for at least 30 minutes.

Each Measure-iT Lead and Cadmium Assay Kit contains:

• Measure-iT Leadmium reagent
• Concentrated Measure-iT Leadmium buffer
• Lead standard
• Cadmium standard
• Detailed protocol (Measure-iT Lead or Cadmium Assay Kit)

Sufficient reagents are provided for 1000 assays using a fluorescence microplate reader.

Phen Green FL and Phen Green SK Indicators

Our phenanthroline-based Phen Green FL and Phen Green SK indicators (P14312), which can be passively loaded into cells as their membrane-permeant diacetates (P6763, P14313), are excellent general-purpose heavy metal sensors that are capable of detecting a broad range of metal ions—including Cu²⁺, Cu⁺ and Fe²⁺ (see above)—as well as micromolar concentrations of Hg²⁺, Pb²⁺, Cd²⁺, Zn²⁺ and Ni²⁺ (Figure 19.7.4). We have used these versatile sensors to detect metal ions in a variety of matrices, including seawater and various contaminated solids such as paint sludge and soil. In such samples, Phen Green FL and Phen Green SK detect only the readily soluble (bioavailable) fraction of the total metal ions. Phen Green FL and Phen Green SK are well suited for initial field testing of metal ion contamination in aqueous samples; the large fluorescence changes produced by micromolar ion concentrations of these heavy metals are easily visible upon illuminating the sample with a hand-held light source (Figure 19.7.1).

Traditional Ca²⁺ and Mg²⁺ Indicators as Pb²⁺, Cd²⁺ and Hg²⁺ Indicators

The fluorescence of several of our traditional Ca²⁺ and Mg²⁺ indicators is strongly affected by binding of Pb²⁺, Cd²⁺ and Hg²⁺ (Figure 19.7.2). Fura-2 and quin-2 bind Cd²⁺ with extremely high affinity (Response of fura-2 and indo-1 to some divalent cations other than Ca2+ and Mg2+—Table 19.5). The excitation response of fura-2 to Cd²⁺—almost identical to its Ca²⁺ response—has been used to monitor Cd²⁺ uptake by cells and to image intracellular free Cd²⁺. The response is reversed by TPEN, which complexes many heavy metals but not Ca²⁺ or Mg²⁺. Heavy metal binding by rhod-5N (R14207, Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) has also been reported; the K_d(Cd²⁺) of rhod-5N was determined to be 1.4 nM.

Mag-fura-2 AM (M1292; Fluorescent Mg2+ Indicators—Section 19.6) has proven useful as an intracellular Cd²⁺ indicator, and indo-1 AM can be used to simultaneously determine the intracellular concentrations of Ca²⁺ and Cd²⁺ or Ca²⁺ and Ba²⁺. Use of fura-2 AM (F1201, F1221, F1225, F14185; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2) to measure the concentration of cytosolic Ba²⁺ has also been reported, as has fluorometric detection of Cd²⁺ at pH 13.3 using calcein (C481). Pb²⁺ entry into cells has been monitored using fura-2 or indo-1 as intracellular indicators. Pb²⁺ is efficiently transported into cells with high selectivity by ionomycin (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). ¹⁹F NMR can also be used to detect Pb²⁺ uptake by platelets using fluorinated BAPTA derivatives (Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8).

Newport Green Indicators

Much of the recent interest in Ni²⁺ comes from its use in the detection and isolation of oligohistidine fusion proteins by metal-chelate affinity chromatography. Newport Green DCF indicator is an exceptionally sensitive probe for Ni²⁺ in solution. 100 µM Ni²⁺ enhances the fluorescence of the Newport Green DCF reagent approximately 13-fold without a spectral shift; Zn²⁺ and Co²⁺ enhance this indicator's fluorescence to a lesser extent (Figure 19.7.4). Newport Green DCF diacetate (N7991) has been used to measure the cellular uptake of Ni²⁺ in human monocyte–derived dendritic cells. Newport Green DCF and Newport Green DCF diacetate have also been used with flow cytometry to detect Ni²⁺-binding metalloproteins involved in human nickel allergy, the most common form of human contact hypersensitivity.

Traditional Ca²⁺ and Mg²⁺ Indicators as Ni²⁺ and Co²⁺ Indicators

Co²⁺ enhances the fluorescence of the Calcium Green-2 indicator at least 20-fold and strongly quenches fluorescence of the Fura Red indicator (Figure 19.7.2). Co²⁺ and Ni²⁺, as well as Cu²⁺ and Fe³⁺ (Figure 19.7.4), strongly quench the fluorescence of calcein, even at pH 7. Consequently, it should be possible to follow the kinetics of uptake of these ions into cells loaded with calcein AM (C1430, C3099, C3100MP). Note that the 1:1 stoichiometry of calcein–metal binding will require the use of low loading levels of this probe to achieve significant quenching by limited amounts of metal transport. The efficient quenching of calcein fluorescence by Co²⁺ or Ni²⁺ has been used to detect liposome fusion (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Fura-2 has been used to measure low levels of Ni²⁺ and Co²⁺ in solution.

Al³⁺ binding has little effect on the fluorescence of most of the traditional Ca²⁺ and Mg²⁺ indicators. However, Al³⁺ is reported to selectively form a fluorescent complex with calcein (C481) at acidic pH that can be detected with micromolar sensitivity. Quenching of the Al³⁺-calcein complex fluorescence has been used as the basis of a method for fluoride determination, with a detection limit of 0.2 ng/mL.

Newport Green DCF diacetate (N7991) has also been used to detect the uptake and distribution of several metal ions—including Al³⁺ and Ti³⁺—in human monocyte–derived dendritic cells by flow cytometry and confocal microscopy. Although the intensities varied, intracellular Cr³⁺, Mo²⁺, Ni²⁺, Ti⁴⁺ and Zr⁴⁺ also produced a fluorescence response with Newport Green DCF diacetate.

La³⁺ has a strong effect on the fluorescence of indicators designed for detection of Ca²⁺, Mg²⁺ and Zn²⁺ (Figure 19.7.2). For example, fluo-4 fluorescence increases more than 400-fold in the presence of 100 µM La³⁺, a response that is about twice as large as that generated by Ca²⁺ saturation. Detection of La³⁺ by the prototypical BAPTA-based Ca²⁺ indicator quin-2 has been used to investigate the ion transport selectivity of 4-bromo A-23187 and ionomycin (I24222; Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). These indicators generally exhibit relatively weak responses to Tb³⁺; fluo-3 and fluo-4 are apparently the most sensitive (~40-fold fluorescence enhancement with 1 µM Tb³⁺) (Figure 19.7.2). Long-lived luminescence of Tb³⁺ (from TbCl₃) is also used to probe Ca²⁺ binding sites of proteins. Fluo-5N (F14203; Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) is a low-affinity Ca²⁺ indicator with subnanomolar affinity for gadolinium (Gd³⁺), enabling its use for determining Gd³⁺-binding affinities of magnetic resonance imaging (MRI) contrast agents via competition titrations.

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