Not only do transition metal ions play an important role in biological structure and activity,ref but they can also serve as surrogates and blockers of ion transport through Ca2+ channels ref (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.ref Detection methods rely both on novel fluorescent ion sensors specifically designed for metal detection,ref as well as on new applications for indicators originally designed for detection of Ca2+ and Mg2+ ref (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.ref 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 (Kd) 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.ref

Applications of Ca2+ and Mg2+ Indicators for Detection of Zn2+ and Other Metals

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+.ref 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+;ref 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 ref (T1210, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8). 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 ref
  • Discriminating fluorescence signals from mitochondrial and cytosolic indicator populations ref
  • Estimating the fraction of residual Ca2+-insensitive indicators after AM ester loading ref
  • Estimating indicator leakage in measurements on cell suspensions in cuvettesref

Mn2+ is also extensively used as an ionic surrogate for measuring Ca2+ influx through ion channels ref (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,ref although such clarity may be impaired by the existence of alternative Mn2+-influx pathways.ref 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.ref 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 ref and also serves as a blocker of mitochondrial permeability transition pore opening.ref

Fluorescence of calcein (C481) is quenched strongly by Co2+, Ni2+ and Cu2+ and appreciably by Fe3+ and Mn2+ at physiological pH ref (panel A, Figure 19.7.4). This fluorescence quenching response can be exploited for detecting the opening of the mitochondrial permeability transition pore,ref 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.ref

Fuorescent Indicators Metal Ions
Figure 19.7.1
Visual screening of fluorescent indicator responses to metal ions. The image shows a 96-well microplate containing various combinations of ions and indicators in 50 mM MOPS pH 7.0. Each row of wells represents a different indicator; each column of wells represents a different ion. The indicators (top to bottom) are: Calcium Green-5N (0.5 µM), Calcium Green-2 (0.2 µM), fluo-4 (2 µM), fluo-5N (2 µM), FluoZin-1 (2 µM), Newport Green DCF (2 µM), Phen Green FL (2 µM; this indicator salt is no longer routinely available, please inquire) and calcein (0.5 µM). The left-hand column of wells contains 10 mM EGTA + 10 µM TPEN (ion-free reference solution). Subsequent columns (left to right) represent 1 µM concentrations of Ca2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, and Pb2+, respectively. The microplate was scanned using a FLA3000G laser scanner (Fuji Photo Film Co.) with excitation at 473 nm and fluorescence emission detected at 520 nm. The image is pseudocolored according to fluorescence intensity (high = red > orange > yellow > green > blue = low).

Figure 19.7.2 Metal-ion response screening for various fluorescent indicators. The maximum relative fluorescence intensity was measured for identical indicator concentrations in solutions containing 10 mM EGTA + 10 µM TPEN, 1 µM ion (100 µM for Mg2+) and 100 µM ion (10 mM for Mg2+). Results are plotted as fluorescence changes relative to the ion-free (10 mM EGTA + 10 µM TPEN) reference solution expressed as (F–Fo)/Fo, where F is the fluorescence intensity of ion-containing solutions and Fo is the fluorescence intensity of the reference solution. Blue bars indicate the response to 1 µM ion (100 µM for Mg2+), and red bars indicate the response to 100 µM ion (10 mM for Mg2+). The indicators represented are: A) Calcium Green-1, B) Calcium Green-2, C) Calcium Green-5N, D) Magnesium Green, E) fluo-3, F) fluo-4, G) fluo-5N, H) FluoZin-1, I) rhod-2, J) rhod-5N, K) X-rhod-1, L) fura-2, M) mag-fura-2, N) FuraZin-1 (no longer routinely available; please inquire), O) Fura Red, P) fura-FF, Q) indo-1, R) FluoZin-3 and S) BTC.

ATP intracellular calcium
Figure 19.7.3
Discrimination of ATP-induced intracellular Ca2+ release from capacitative Ca2+ entry in bovine vascular endothelial cells using quenching of fura-2 fluorescence by Mn2+. Intracellular Ca2+ release was detected by fura-2 fluorescence excited alternately at 360 nm and 380 nm (F360/F380). Capacitative entry of extracellular Mn2+ was detected by its quenching effect on the Ca2+–insensitive fura-2 fluorescence signal excited at 360 nm (F360). Successive Ca2+ spikes induced by sustained applications of low ATP concentrations (panel A) or transient 5-second applications of 2 µM ATP (panel B) were followed by accelerated entry of Mn2+. Experiments were conducted in Ca2+-free extracellular medium containing 100 µM Mn2+. Under these conditions, Mn2+ functions as an ionic surrogate for Ca2+ with respect to transmembrane conductance, and Ca2+ signals can be unambiguously attributed to release from intracellular stores. Figure reproduced with permission from ref.

Screening of calcium
Figure 19.7.4 Metal-ion response screening for calcein, Newport Green and Phen Green indicators. The maximum relative fluorescence intensity was measured for identical indicator concentrations in solutions containing 10 mM EGTA + 10 µM TPEN, 1 µM ion and 100 µM ion. Results are plotted as fluorescence changes relative to the ion-free (10 mM EGTA + 10 µM TPEN) reference solution expressed as (F–Fo)/Fo, where F is the fluorescence intensity of ion-containing solutions and Fo is the fluorescence intensity of the reference solution. Blue bars indicate the response to 1 µM ion, and red bars indicate the response to 100 µM ion. The indicators represented are: A) calcein, B) Newport Green DCF, C) Newport Green PDX, D) Phen Green FL and E) Phen Green SK. The water-soluble salt forms of the Newport Green PDX and Phen Green FL indicators are no longer routinely available; please inquire.

Indicators for Zinc

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 ref and is a contributory factor in neurological disorders including epilepsy and Alzheimer disease.ref Free Zn2+ is released from metalloprotein complexes during oxidative stress.ref The intracellular concentration of free Zn2+ is extremely low in most cells (<1 nM), with the remainder being bound to proteins or nucleic acids.ref One calculation ref 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.ref

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.ref 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.ref Peak concentrations of synaptically released Zn2+ may exceed 100 µM. We have developed FluoZin-1 (F24180, F24181) 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 ref (panel H, Figure 19.7.2; Figure 19.7.5). In our laboratories, we determined a Kd(Zn2+) of 8.2 µM for FluoZin-1; however, dissociation constants are known to vary considerably depending on the experimental conditions and a Kd(Zn2+) of 0.4 µM for this same indicator has been reported elsewhere,ref 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.ref

The FluoZin-3 indicator ref (F24194, structure; F24195) is a Zn2+-selective indicator with a structure that resembles fluo-4. FluoZin-3 exhibits high Zn2+-binding affinity (Kd for Zn2+ ~15 nM) that is unperturbed by Ca2+ concentrations up to at least 1 µM.ref 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.ref 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.ref Although imaging applications of FluoZin-3 are predominant, cell-based microplate assays ref and flow cytometry protocols ref have also been developed.

Fluorescence Fluozin

Figure 19.7.5 Zn2+ dependence of the fluorescence emission spectra of FluoZin-2.

Fluorescence Fluozin-3

Figure 19.7.6 Zn2+ dependence of the fluorescence emission spectra of FluoZin-3 (F24194). The spectrum for the Zn2+-free solution is indistinguishable from the baseline.

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 Kd for Zn2+ of ~65 nM.ref The cell-permeant AM ester form of RhodZin-3 (R36351) effectively localizes into mitochondria and is a valuable tool for investigating the physiological consequences of mitochondrial Zn2+ sequestration.ref The cell-impermeant RhodZin-3 salt (R36350) can be used to measure extracellular Zn2+ concentrations.ref

Newport Green DCF and Newport Green PDX Indicators

The Newport Green DCF indicator (N7990, structure; N7991) has moderate zinc-binding affinity (Kd for Zn2+ ~1 µM) but is essentially insensitive to Ca2+ (Kd for Ca2+ >100 µM), making this a valuable probe for detecting Zn2+ influx into neurons through voltage- or glutamate-gated channels.ref 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.ref Newport Green DCF has been used to identify insulin-producing β-cells from human pancreatic islets based on their high intracellular Zn2+ content ref (photo). 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.ref The response of Newport Green DCF to transition metals, including Zn, Mn, Fe, Co, Cu(I), Cu(II), Ni and Cd, have been characterized.ref

Newport Green PDX ref (N24191) incorporates the same di-(2-picolyl)amine chelator as Newport Green DCF but has a higher Zn2+ dissociation constant (Kd for Zn2+ ~30 µM) and a larger Zn2+-free to Zn2+-saturated fluorescence intensity increase.


Use of the membrane-permeant probe N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ, M688; structure) in cells was first described by Fredrickson.ref 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,ref but a 1:1 complex may be formed with metalloproteins. The intracellular Zn2+ chelator dithizone blocks TSQ binding of Zn2+.ref

Several reports suggest that TSQ can be used to localize Zn2+ pools in the central nervous system.ref 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.ref 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.ref 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.ref

TSQ-based assays for Zn2+ in seawater and other biological systems exhibit a detection limit of ~0.1 nM.ref The simultaneous determination of Zn2+ and Cd2+ by spectrofluorometry using TSQ in an SDS micelle has also been reported.ref TSQ has been used to measure Zn2+ levels in artificial lipid vesicles and live sperm cells by flow cytometry.ref 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+.ref For example, fura-2 (F1200, F6799; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2) exhibits a Kd 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 ref and for examining Zn2+ levels in live neurons ref and myocytes.ref Fura-2 has also been used as an indicator for nanomolar levels of free Zn2+ and other ions in solution.ref

Mag-fura-2 (M1290, Fluorescent Mg2+ Indicators—Section 19.6) exhibits slightly altered spectral characteristics upon binding Zn2+ (Kd ~20 nM at pH 7.0–7.8 and 37°C), allowing Zn2+ to be measured in the presence of Ca2+.ref A review by Dineley and co-workers ref 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 (Kd) 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 Kd for Zn2+ around 20 nM, appear to be the most suitable.ref Based on their similar ion-binding properties, the Magnesium Green indicator (M3733, Fluorescent Mg2+ Indicators—Section 19.6) and our mag-fluo-4 indicator (M14205, Fluorescent Mg2+ Indicators—Section 19.6) are suitable for confocal imaging of Zn2+ influx.ref

Indicators for Copper

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.ref 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.ref

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+.ref 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.ref

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+.ref 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 ref (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.ref 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 ref (Kd = 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+.ref

Indicators for Iron

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.ref Fluorescence of Phen Green FL (available only as the cell-permeant diacetate, P6763) and Phen Green SK (P14312, structure; 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.ref Phen Green SK diacetate has been successfully used to quantitate the intracellular pool of chelatable iron in rat hepatocytes,ref to directly measure Fe2+ transport across chloroplast membranes ref and to monitor iron uptake in relation to fatigue in mouse skeletal muscle.ref


Cabantchik and co-workers have exploited the fluorescence quenching of calcein at neutral pH to follow intracellular release of Fe2+ from transferrin.ref 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+ ref (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.ref 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.ref

Indicators for Lead, Cadmium and Mercury

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.ref



Figure 19.7.7 Dual-color scatter plot showing two populations of Jurkat cells. Jurkat cells were loaded with Leadmium Green AM (A10024) and washed. The sample was then incubated in the presence of 1 µM PbCl2 (in saline) and 1 µM ionomycin. After washing the sample, it was incubated in the presence of PI. Dual-color fluorescence was collected using 488 nm excitation and 525/10 nm and 610/10 nm bandpass filters. This results in visualization of two populations: dead cells (positive for PI) and live cells that contain lead (positive for Leadmium Green dye and negative for PI).

Measure-iT Lead and Cadmium Assay Kit

The Measure-iT Lead and Cadmium Assay Kit (M36353) provides a fluorescence microplate assay for quantitation of lead or cadmium in solution, with a linear detection range from 5 to 200 nM.ref (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:

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



Figure 19.7.8 Linearity and sensitivity of the Measure-iT Lead and Cadmium Assay Kit (M36353) for lead (Panel A) and cadmium (Panel B). Triplicate 10 µL samples of lead and cadmium were assayed; fluorescence was measured at 490/520 nm and plotted versus lead or cadmium concentration. The variation (CV) of replicate samples was <2%.

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+.ref The response is reversed by TPEN (T1210, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), which complexes many heavy metals but not Ca2+ or Mg2+.ref Heavy metal binding by rhod-5N (R14207, Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) has also been reported; the Kd(Cd2+) of rhod-5N was determined to be 1.4 nM.ref

Mag-fura-2 AM (M1291, M1292; Fluorescent Mg2+ Indicators—Section 19.6) has proven useful as an intracellular Cd2+ indicator,ref and indo-1 AM can be used to simultaneously determine the intracellular concentrations of Ca2+ and Cd2+ or Ca2+ and Ba2+.ref 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,ref as has fluorometric detection of Cd2+ at pH 13.3 using calcein ref (C481). Pb2+ entry into cells has been monitored using fura-2 or indo-1 as intracellular indicators.ref Pb2+ is efficiently transported into cells with high selectivity by ionomycin ref (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 ref (Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8).

Indicators for Nickel and Cobalt

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 (N7990) is an exceptionally sensitive probe for Ni2+ in solution.ref 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.ref 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.ref The AM ester of Newport Green PDX (N24191) incorporates the same di-(2-picolyl)amine chelator as Newport Green DCF and should prove to have similar utility for detection of Ni2+.

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.ref 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+ ref has been used to detect liposome fusion ref (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.ref

Indicators for Aluminum

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.ref 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.ref

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.ref Although the intensities varied, intracellular Cr3+, Mo2+, Ni2+, Ti4+ and Zr4+ also produced a fluorescence response with Newport Green DCF diacetate.

Indicators for Lanthanides

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 ref (B1494, 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, T1247) is also used to probe Ca2+ binding sites of proteins.ref 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.ref

Data Table

         High Zn2+            Low Zn2+      
Cat # Links MW Storage Soluble Abs EC Em Solvent Abs EC Em Solvent Product Kd Notes
C481 icon icon 622.54 L pH >5 494 77,000 517 pH 9     see Notes     see Notes 1, 2
C1430 icon icon 994.87 F,D DMSO <300   none           C481    
C3099 icon icon 994.87 F,D DMSO <300   none           C481   3
C3100MP icon icon 994.87 F,D DMSO <300   none           C481    
F24180 icon 599.67 F,D,L pH >6 490 68,000 see Notes H2O 495 58,000 517 H2O/Zn2+   8.2 µM 4, 5, 6, 7
F24181 icon 701.59 F,D,L DMSO 456 26,000 see Notes MeOH         F24180   8
FluoZin-2 icon 800.89 D,L pH >6 494 74,000 522 H2O 494 75,000 521 H2O/Zn2+   2.0 µM 4, 5, 6
F24189 icon 876.73 F,D,L DMSO 299 16,000 none MeOH         FluoZin-2    
F24194 icon 846.96 F,D,L pH >6 491 82,000 see Notes H2O 494 88,000 516 H2O/Zn2+   15 nM 4, 5, 6, 7, 9
F24195 icon 982.85 F,D,L DMSO 455 26,000 see Notes MeOH         F24194   8
M688 icon 328.38 L EtOH 334 4200 385 MeOH     see Notes       10
N7990 icon 793.74 D,L pH >6 506 82,000 535 H2O 506 82,000 535 H2O/Zn2+   1.0 µM 4, 5, 6
N7991 icon 801.64 F,D DMSO 302 15,000 none MeOH         N7990    
Newport Green PDX icon 521.52 D,L pH >6 490 76,000 518 H2O 491 77,000 518 H2O/Zn2+   30 µM 4, 5, 6
N24191 icon 593.59 F,D DMSO 457 21,000 538 MeOH         Newport Green PDX    
P6763 icon 668.68 F,D DMSO <300   none           Phen Green FL    
Phen Green FL icon 660.79 D,L pH >6 492 68,000 517 pH 9     see Notes       11
P14312 icon 698.60 D,L pH >6 507 86,000 532 pH 9     see Notes       11
P14313 icon 706.49 F,D DMSO <300   none           P14312    
R36350 icon 788.94 F,D,L pH >6 549 80,000 see Notes H2O 552 82,000 576 H2O/Zn2+   65 nM 4, 5, 6, 7, 9
R36351 icon 1009.86 F,D,L DMSO 546 110,000 570 see Notes         R36350   12
T1247 icon 373.38 D H2O 270 4700 545 H2O             13, 14
  1. C481 fluorescence is strongly quenched by micromolar concentrations of Fe3+, Co2+, Ni2+ and Cu2+ at pH 7.ref
  2. Kd(Co2+) for calcein is 120 nM, determined in 10 mM HEPES, 1 µM Ca2+, 1 mM Mg2+, 100 mM KCl.ref
  3. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
  4. Dissociation constants are known to vary considerably depending on the temperature, pH, ionic strength, viscosity, protein binding, presence of other ions (especially polyvalent ions), instrument setup and other factors. It is strongly recommended that these values be verified under user-specific experimental conditions.
  5. Spectra measured in aqueous buffers containing zero (H2O) or a >10-fold excess of free cation X (H2O/X) relative to the listed dissociation constant (Kd) for cation X.
  6. This indicator exhibits fluorescence enhancement in response to ion binding, with essentially no change in absorption or emission wavelengths.
  7. Fluorescence of the free indicator is very weak and is enhanced >100-fold on binding Zn2+.
  8. Fluorescence of this AM ester derivative is very weak and is enhanced only after hydrolytic cleavage followed by binding of divalent cations to the anionic indicator.
  9. Kd(Zn2+) determined in 135 mM NaCl, 1.1 mM EGTA, 20 mM HEPES pH 7.4, 0 to 10 µM free Zn2+ at 22°C. Kd(Zn2+) is unchanged in the presence of 1 µM free Ca2+.ref
  10. Fluorescence of M688 is very sensitive to solvent polarity. Em = 495 nm in aqueous buffer (pH 7.4). Fluorescence is enhanced on binding Zn2+ (0.01–10 µM) with no change in emission wavelength.ref
  11. Fluorescence of Phen Green indicators is quenched by Fe2+, Fe3+, Cu2+, Cd2+, Hg2+ and Pb2+ with no change in emission wavelength. The extent of quenching is dependent on the concentration of indicator as well as the ion concentration.
  12. Spectra measured in 90:10 (v/v) CHCl3:MeOH.
  13. Absorption and luminescence of T1247 are extremely weak unless it is chelated. Data are for dipicolinic acid (DPA) chelate. The luminescence spectrum has secondary peak at 490 nm.
  14. MW is for the hydrated form of this product.