Related Technical Notes
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This section describes fluorescent indicators for intracellular and extracellular chloride together with an assortment of analytical reagents and methods for direct or indirect quantitation of other inorganic anions, including bromide, iodide, hypochlorite, cyanide,nitrite, nitrate, phosphate, pyrophosphate and selenide.
Most of the fluorescent chloride indicators are 6-methoxyquinolinium derivatives, the prototype of which is 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cl– detection sensitivity has been improved by modifications of the quinolinium N substituent. Cl– indicators include:
All of these indicators detect Cl– via diffusion-limited collisional quenching. This detection mechanism is different from that of fluorescent indicators for Ca2+, Mg2+, Zn2+, Na+ and K+. It involves a transient interaction between the excited state of the fluorophore and a halide ion—no ground-state complex is formed. Quenching is not accompanied by spectral shifts (Figure 21.2.1) and, consequently, ratio measurements are not directly feasible. Quenching by other halides, such as Br– and I–, and other anions, such as thiocyanate, is more efficient than Cl– quenching. Fortunately, physiological concentrations of non-choloride ions do not significantly affect the fluorescence of SPQ and other methoxyquinolinium-based Cl– indicators. With some exceptions, fluorescence of these indicators is not pH sensitive in the physiological range. Because Cl–-dependent fluorescence quenching is a diffusional process, it is quite sensitive to solution viscosity and volume. Exploiting this property, SPQ has been used to measure intracellular volume changes.
The efficiency of collisional quenching is characterized by the Stern–Volmer constant (KSV), defined as the reciprocal of the ion concentration that produces 50% of maximum quenching. For SPQ, KSV is reported to be 118 M-1 in aqueous solution and 12 M-1 inside cells. For MQAE, in situ KSV values of 25–28 M-1 have been determined in various cell types, compared with the solution value of 200 M-1. Intracellular Cl– indicators are generally calibrated using high-K+ buffers and the K+/H+ ionophore nigericin (N1495) in conjunction with tributyltin chloride, an organometallic compound that acts as a Cl–/OH– antiporter. With the exception of diH-MEQ (see below), Cl– indicators must be loaded into cells by long-term incubation (up to eight hours) in the presence of a large excess of dye or by brief hypotonic permeabilization. Because membranes are slightly permeable to the indicator, rapid leakage may occur. Experimentally determined estimates of leakage vary quite widely.
Measurement of intracellular Cl– concentrations and the study of Cl– channels have been stimulated by the discovery that cystic fibrosis is caused by mutations in a gene encoding a Cl– transport channel, which is known as the cystic fibrosis transmembrane conductance regulator (CFTR). Cl– permeability assays are used to detect activity of the CFTR and other anion transporters. In these assays, SPQ- or MQAE-loaded cells are successively perfused with chloride-containing extracellular medium followed by medium in which the Cl– content is replaced by nitrate (NO3–). NO3– is used in this assay protocol because it produces no fluorescence quenching of the indicator, yet its channel permeability is essentially the same as that of Cl– (Figure 21.2.2).
Figure 21.2.2 Detection of cystic fibrosis transmembrane conductance regulator (CFTR) activity using 6-methoxy-N-(3-sulfopropyl)quinolinium, inner salt (SPQ, M440). Fluorescence of intracellular SPQ is quenched by collision with chloride ions, indicated by F0/F > 1 (F0 = fluorescence intensity in absence of chloride, F = fluorescence intensity at time points indicated on the x-axis). Upon addition of cyclic AMP to initiate channel opening, and exchange of extracellular Cl– (135 mM) for nitrate (NO3–), SPQ quenching decreases in CFTR-expressing cells (filled circles) as CFTR-mediated anion transport results in replacement of intracellular Cl– with nonquenching NO3–. Control cells with no CFTR expression (open circles) show no response.
SPQ (M440) is currently in widespread use for detecting CFTR activity using the Cl–/NO3– exchange technique described above. SPQ has also has been employed to investigate Cl– fluxes through several other transporters such as the GABAA receptor, erythrocyte Cl–/HCO3– exchangers and the mitochondrial uncoupling protein. Although SPQ requires UV excitation (as do MQAE and MEQ), techniques for flow cytometric detection and calibration of the indicator using argon-ion laser excitation at 351 nm and 364 nm have been successfully demonstrated.
MQAE (E3101) has greater sensitivity to Cl– and a higher fluorescence quantum yield than SPQ; consequently, it is currently the more widely used of the two indicators. However, the ester group of MQAE may slowly hydrolyze inside cells, resulting in a change in its fluorescence response. MQAE has been used in a fluorescence-based microplate assay that has potential for screening compounds that modify Cl– ion-channel activity. Other applications have included Cl– measurements in cytomegalovirus-infected fibroblasts, smooth muscle cells and salivary glands, as well as in reconstituted membranes containing the GABAA receptor or the mitochondrial-uncoupling protein (UCP-1).
The Cl– indicator 6-methoxy-N-ethylquinolinium iodide (MEQ) can be rendered cell-permeant by masking its positively charged nitrogen to create a lipophilic, Cl–-insensitive compound, 6-methoxy-N-ethyl-1,2-dihydroquinoline (dihydro-MEQ). This reduced quinoline derivative can then be loaded noninvasively into cells, where it is rapidly reoxidized in most cells to the cell-impermeant, Cl–-sensitive MEQ (Figure 21.2.3). Using this technique, researchers have loaded live brain slices and hippocampal neurons with MEQ for confocal imaging of Cl– responses to GABAA receptor activation and glutamatergic excitotoxicity. Quenching of intracellular MEQ fluorescence by Cl– has a KSV of 19 M-1, a value that is slightly higher than that reported for SPQ in fibroblasts. MEQ is often supplied in solid form that can be reduced with a simple protocol (6-Methoxy-N-ethylquinolinium Iodide) to dihydro-MEQ with sodium borohydride just prior to cell loading.
Figure 21.2.3 Intracellular delivery of the fluorescent chloride indicator 6-methoxy-N-ethylquinolinium iodide (MEQ) via oxidation of the membrane-permeant precursor dihydro-MEQ.
The fluorescence of lucigenin is quantitatively quenched by high levels of Cl– with a reported KSV = 390 M-1. Lucigenin absorbs maximally at both 368 nm (ε = 36,000 cm-1M-1) and 455 nm (ε = 7400 cm-1M-1), with an emission maximum at 505 nm. Its fluorescence emission has a quantum yield of ~0.6 and is insensitive to nitrate, phosphate and sulfate. Lucigenin is a useful Cl– indicator in liposomes and reconstituted membrane vesicles; however, because its fluorescence is reported to be unstable in the cytoplasm, it may not always be suitable for determining intracellular Cl–. Lucigenin has been used to detect chloride uptake in tonoplast vesicles and to measure Cl– influx across the pleural surface in perfused mouse lungs.
As mentioned above, the fluorescence of SPQ and related Cl– indicators is quenched by collision with a variety of anions, including (in order of increasing quenching efficiency) Cl–, Br–, I– and thiocyanate (SCN–). For example, fluorescence of SPQ is partially quenched by the anionic pH buffer TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) but not by the protonated TES zwitterion, a property that has been exploited to measure proton efflux from proteoliposomes. Anion detectability using diffusional fluorescence quenching of these fluorophores is typically limited to the millimolar range. I– quenches many other fluorophores and is commonly used to determine the accessibility of fluorophores to quenching in proteins and membranes.
In addition, halides can be oxidized to hypohalites (–OCl, –OBr, –OI), which react with rhodamine 6G (Probes for Mitochondria—Section 12.2) to yield chemiluminescent products. A cell produces –OCl by oxidizing Cl– within the phagovacuole. –OCl also reacts with fluorescein (F1300, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5) to yield fluorescent products, permitting analysis of –OCl levels in water.
Alternatively, 3'-(p-aminophenyl) fluorescein (APF) and 3'-(p-hydroxyphenyl) fluorescein (HPF) (A36003, H36004; Generating and Detecting Reactive Oxygen Species—Section 18.2) can be used for the selective detection of –OCl. Both of these fluorescein derivatives are essentially nonfluorescent until they react with the hydroxyl radical (HO·) or peroxynitrite anion (ONOO–) (Figure 21.2.4). APF will also react with the hypochlorite anion (–OCl), making it possible to use APF and HPF together to selectively detect hypochlorite anion. In the presence of these specific ROS, both APF and HPF yield a bright green-fluorescent product (excitation/emission maxima ~490/515 nm) and are compatible with all fluorescence instrumentation capable of visualizing fluorescein. Using APF, researchers have been able to detect the –OCl generated by activated neutrophils, a feat that has not been possible with traditional ROS indicators.
The fluorescent protein–based Premo Halide Sensor (P10229) is a pharmacologically relevant sensor for functional studies of ligand- and voltage-gated chloride channels and their modulators in cells. Chloride channels are involved in cellular processes as critical and diverse as transepithelial ion transport, electrical excitability, cell volume regulation and ion homeostasis. Given their physiological significance, it follows that defects in their activity can have severe implications, including such conditions as cystic fibrosis and neuronal degeneration. Thus, chloride channels represent important targets for drug discovery.
Premo Halide Sensor combines a yellow-fluorescent protein (YFP) variant sensitive to halide ions with the efficient and noncytopathic BacMam delivery and expression technology (BacMam Gene Delivery and Expression Technology—Note 11.1), yielding a highly sensitive, robust and easy-to-use tool for efficiently screening halide ion channels and transporter modulators in their cellular models of choice. Premo Halide Sensor is based on the Venus variant of Aequorea Victoria green-fluorescent protein (GFP), which displays enhanced fluorescence, increased folding, and reduced maturation time when compared with YFP. Additional mutations H148Q and I152L were made within the Venus sequence to increase the sensitivity of the Venus fluorescent protein to changes in local halide concentration, in particular iodide ions. Because chloride channels are also permeable to the iodide ion (I), iodide can be used as a surrogate of chloride. Upon stimulation, a chloride channel or transporter opens and iodide flows down the concentration gradient into the cells, where it quenches the fluorescence of the expressed Premo Halide Sensor protein (Figure 21.2.5, Figure 21.2.6). The decrease in Premo Halide Sensor fluorescence is directly proportional to the ion flux, and therefore the chloride channel or transporter activity. Premo Halide Sensor shows a similar excitation and emission profile to YFP (Figure 21.2.7) and can be detected using standard GFP/FITC or YFP filter sets. Halide-sensitive YFP-based constructs in conjunction with iodide quenching have been used in high-throughput screening (HTS) to identify modulators of calcium-activated chloride channels.
Premo Halide Sensor (P10229) is pre-packaged and ready for immediate use. It contains all components required for cellular delivery and expression—including baculovirus carrying the genetically encoded biosensor, BacMam enhancer and stimulus buffer—in ten 96- or 384-well plates. Premo Halide Sensor has been demonstrated to transduce multiple cell lines including BHK, U2OS, HeLa, CHO, and primary human bronchial epithelial cells (HBEC), providing the flexibility to assay chloride permeable channels in a wide range of cellular models.
The homologous aromatic dialdehydes, o-phthaldialdehyde (OPA) and naphthalene-2,3-dicarboxaldehyde (NDA, N1138), are essentially nonfluorescent until reacted with a primary amine in the presence of excess cyanide or a thiol, such as 2-mercaptoethanol, 3-mercaptopropionic acid or the less obnoxious sulfite, to yield a fluorescent isoindole (Figure 21.2.8, Figure 21.2.9). Modified protocols that use an excess of an amine and limiting amounts of other nucleophiles permit the determination of cyanide in blood, urine and other samples.
We also offer the ATTO-TAG CBQCA (A6222) and ATTO-TAG FQ (A10192) reagents, which are similar to OPA and NDA in that they react with primary amines in the presence of cyanide or thiols to form highly fluorescent isoindoles (Figure 21.2.10). The ATTO-TAG CBQCA and ATTO-TAG FQ reagents should also be useful for detecting cyanide in a variety of biological samples.
We have found that our Thiol and Sulfide Quantitation Kit (T6060, Introduction to Thiol Modification and Detection—Section 2.1) also provides an ultrasensitive enzymatic assay for cyanide, with a detection limit of ~5 nanomoles. In this case, interference would be expected from thiols, sulfides, sulfites and other reducing agents.
The PiPer Phosphate Assay Kit (P22061) provides an ultrasensitive assay that detects free phosphate in solution through formation of the fluorescent product resorufin. Because resorufin also has strong absorption, the assay can be performed either fluorometrically or spectrophotometrically. This kit can be used to detect inorganic phosphate (Pi) in a variety of samples or to monitor the kinetics of phosphate release by a variety of enzymes, including ATPases, GTPases, 5'-nucleotidase, protein phosphatases, acid and alkaline phosphatases and phosphorylase kinase. Furthermore, the assay can be modified to detect virtually any naturally occurring organic phosphate molecule by including an enzyme that can specifically digest the organic phosphate to liberate inorganic phosphate.
In the PiPer phosphate assay (Figure 21.2.11), maltose phosphorylase converts maltose (in the presence of Pi) to glucose 1-phosphate and glucose. Then glucose oxidase converts the glucose to gluconolactone and H2O2. Finally, with horseradish peroxidase as a catalyst, the H2O2 reacts with the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) to generate resorufin, which has absorption/emission maxima of ~571/585 nm. The resulting increase in fluorescence or absorption is proportional to the amount of Pi in the sample. This kit can be used to detect as little as 0.2 µM Pi by fluorescence (Figure 21.2.12) or 0.4 µM Pi by absorption.
The PiPer Phosphate Assay Kit contains:
Each kit provides sufficient reagents for approximately 1000 assays using a reaction volume of 100 µL per assay and either a fluorescence or absorbance microplate reader.
Figure 21.2.12 Detection of inorganic phosphate using the PiPer Phosphate Assay Kit (P22061). Each reaction contained 50 µM Amplex Red reagent, 2 U/mL maltose phosphorylase, 1 mM maltose, 1 U/mL glucose oxidase and 0.2 U/mL HRP in 1X reaction buffer. Reactions were incubated at 37°C. After 60 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Data points represent the average of duplicate reactions, and a background value of 43 (arbitrary units) was subtracted from each reading.
tThe PiPer Pyrophosphate Assay Kit (P22062) provides a sensitive fluorometric or colorimetric method for measuring the inorganic pyrophosphate (PPi) in experimental samples or for monitoring the kinetics of PPi release by a variety of enzymes, including DNA and RNA polymerases, adenylate cyclase and S-acetyl coenzyme A synthetase. In the PiPer pyrophosphate assay, inorganic pyrophosphatase hydrolyzes PPi to two molecules of inorganic phosphate (Pi). The Pi then enters into the same cascade of reactions as it does in the PiPer Phosphate Assay Kit (Figure 21.2.11). In this case, the resulting increase in fluorescence or absorption is proportional to the amount of PPi in the sample. This kit can be used to detect as little as 0.1 µM PPi by fluorescence or 0.2 µM PPi by absorption (Figure 21.2.13).PiPer Pyrophosphate Assay Kit
The PiPer Pyrophosphate Assay Kit contains:
Each kit provides sufficient reagents for approximately 1000 assays using a reaction volume of 100 µL per assay and either a fluorescence or absorbance microplate reader.
The EnzChek Phosphate Assay Kit (E6646), which is based on a method originally described by Webb, provides an easy enzymatic assay for detecting Pi from multiple sources through formation of a chromophoric product (Figure 21.2.14). Although this kit is usually used to determine the Pi produced by a wide variety of enzymes such as ATPases, kinases and phosphatases (Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3), it can also be used to specifically quantitate Pi with a sensitivity of ~2 µM Pi (~0.2 µg/mL) (Figure 21.2.15). Moreover, this colorimetric assay has proven useful for determining the level of Pi contamination in the presence of high concentrations of acid-labile phosphates using a microplate reader. Because the sulfate anion competes with Pi for binding to purine nucleoside phosphorylase (PNP), this kit can be adapted for measurement of sulfate concentrations between 0.1 and 10 mM in the presence of a low (<100 µM) fixed Pi concentration.
The EnzChek Phosphate Assay Kit contains:
Each kit provides sufficient reagents for about 100 phosphate assays using 1 mL assay volumes and standard cuvettes.
Figure 21.2.15 Quantitative analysis of inorganic phosphate using the EnzChek Phosphate Assay Kit (E6646). KH2PO4 was used as the source for the inorganic phosphate, and the absorbance at 360 nm was corrected for background absorbance. The inset shows an enlargement of the standard curve, demonstrating the lower range of the assay; the units are the same.
In the EnzChek Pyrophosphate Assay Kit (E6645), we have adapted the method provided in the EnzChek Phosphate Assay Kit to permit the sensitive spectrophotometric detection of PPi, which is converted by the enzyme pyrophosphatase to Pi. Because two moles of Pi are released per mole of PPi consumed, the sensitivity limit of the EnzChek Pyrophosphate Assay Kit is 1 µM PPi (~0.2 µg/mL). This assay has been modified to continuously detect several enzymes that liberate PPi such as aminoacyl-tRNA synthetase, luciferase, cytidylyl transferase and S-acetyl coenzyme A synthetase and potentially DNA and RNA polymerases, adenylate cyclase and guanylyl cyclase.
The EnzChek Pyrophosphate Assay Kit contains:
Each kit provides sufficient reagents for about 100 PPi assays using standard 1 mL assay volumes and standard cuvettes.
With the discovery of the role of nitric oxide in signal transduction (Probes for Nitric Oxide Research—Section 18.3), assays for nitrite (NO2–) have assumed new importance. Because inorganic nitrite is spontaneously produced by air oxidation of nitric oxide, the same reagents that have been utilized to detect nitric oxide production in cells should be useful for detecting nitrite in aqueous samples. Furthermore, inorganic nitrate (NO3–) can be reduced to NO2– by both chemical and enzymatic means, permitting the quantitative analysis of NO3– in samples.
The Measure-iT High-Sensitivity Nitrite Assay Kit (M36051) provides an easy and accurate method for quantitating nitrite. This kit has an optimal range of 20–500 picomoles nitrite (Figure 21.2.16) , making it up to 50 times more sensitive than colorimetric methods utilizing the Griess reagent. Nitrates may be analyzed after quantitative conversion to nitrites through enzymatic reduction.
Each Measure-iT High-Sensitivity Nitrite Assay Kit contains:
Simply dilute the reagent 1:100, load 100 µL into the wells of a microplate, add 1–10 µL sample volumes and mix. After a 10-minute incubation at room temperature, add 5 µL of developer and read the fluorescence. The assay signal is stable for at least 3 hours, and common contaminants are well tolerated in the assay. The Measure-iT High-Sensitivity Nitrite Assay Kit provides sufficient material for 2000 assays, based on a 100 µL assay volume in a 96-well microplate format; this nitrite assay can also be adapted for use in cuvettes or 384-well microplates.
Figure 21.2.16 Linearity and sensitivity of the Measure-iT high-sensitivity nitrite assay. Triplicate 10 µL samples of nitrite were assayed using the Measure-iT High-Sensitivity Nitrite Assay Kit (M36051). Fluorescence was measured using excitation/emission of 365/450 nm and plotted versus picomoles of nitrite. Background fluorescence was not subtracted. The variation (CV) of replicate samples was <2%.
Under physiological conditions, NO is readily oxidized to NO2– and NO3– or it is trapped by thiols as an S-nitroso adduct. The Griess reagent provides a simple and well-characterized colorimetric assay for nitrites—and nitrates that have been reduced to nitrites—with a detection limit of about 100 nM. The Griess assay is suitable for measuring the activity of nitrate reductase in a microplate. Nitrite reacts with the Griess reagent to form a purple azo derivative that can be monitored by absorbance at 548 nm (Figure 21.2.17).
The Griess Reagent Kit (G7921) contains all of the reagents required for NO2– quantitation, including:
Both the N-(1-naphthyl)ethylenediamine dihydrochloride and the sulfanilic acid in 5% H3PO4 are provided in convenient dropper bottles for easy preparation of the Griess reagent. Sample pretreatment with nitrate reductase and glucose 6-phosphate dehydrogenase is reported to reduce NO3– without producing excess NADPH, which can interfere with the Griess reaction. NO that has been trapped as an S-nitroso derivative can also be analyzed with the Griess Reagent Kit after first releasing the NO from its complex using mercuric chloride or copper (II) acetate.
DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein, D23841) and its diacetate derivative (DAF-FM diacetate, D23842, D23844; Probes for Nitric Oxide Research—Section 18.3) have significant utility for measuring nitric oxide and nitrite production in live cells and solutions. The fluorescence quantum yield of DAF-FM is reported to be 0.005 but increases about 160 fold to 0.81 after reacting with nitrite (Figure 21.2.18). DAF-FM has some important advantages over the similar nitric oxide sensor, DAF-2, and other aromatic diamines:
Because the reaction of DAF-FM with NO requires a preliminary nonspecific oxidation step, it is important to also perform control experiments with nitric oxide synthase inhibitors to confirm the source of the fluorescent species.
In addition, 2,3-diaminonaphthalene reacts with NO2– to form the fluorescent product 1H-naphthotriazole. A rapid, quantitative fluorometric assay that employs 2,3-diaminonaphthalene can reportedly detect from 10 nM to 10 µM NO2–, and is compatible with a 96-well microplate format. Nitrate (NO3–) does not interfere with this assay; however, NO3– can be reduced to NO2– by bacterial nitrate reductase and then detected using the same reagent. A detailed protocol for measuring the stable products of the nitric oxide pathway (NO2– and NO3–) using 2,3-diaminonaphthalene has been published and is shown to be approximately 50 times more sensitive than the Griess assay.
NBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan) has been used to measure NO2– in water. Reaction of NBD methylhydrazine with NO2– in the presence of mineral acids leads to formation of fluorescent products with excitation/emission maxima of ~468/537 nm. This reaction serves as the principle behind a selective fluorogenic method for the determination of NO2– (Figure 21.2.19). The assay is suitable for measurements by absorption or fluorescence spectroscopy or by fluorescence-detected HPLC.
Rhodamine 110 has proven useful in a fluorescence quenching method for determining trace nitrite. This sensitive assay takes advantage of the reaction of the green-fluorescent rhodamine 110 with nitrite at acidic pH to form a nitroso product that exhibits much weaker fluorescence. With a linear range of 1 × 10-8 to 3 × 10-7moles/L and a detection limit of 7 × 10-10 moles/L, this assay has been used to measure nitrite in tap water and lake water without any prior extraction procedures.
Efficient quenching of SPQ or MQAE fluorescence (M440, E3101; see above) by nitrite (but not nitrate) has been used for direct measurement of NO2– transport across erythrocyte membranes and for functional assays of bacterial nitrite extrusion transporters.
For a detailed explanation of column headings, see Definitions of Data Table Contents
|305.29||F,D,L||MeOH||465||ND||560||MeOH||1, 2, 3|
|412.35||F,D,L||DMSO||487||84,000||see Notes||pH 8||6|
|326.19||F,D,L||H2O||350||2800||460||H2O||200 M-1||7, 8, 9, 10|
EnzChek Pyrophosphate assay reagent
|313.33||FF,D||H2O||332||16,000||none||pH 7||11, 12|
EnzChek Phosphate assay reagent
|313.33||F,D||H2O||332||16,000||none||pH 7||11, 12|
|lucigenin||510.50||L||H2O||455||7400||505||H2O||390 M-1||7, 8, 10, 13, 14|
|281.33||L||H2O||344||3700||443||H2O||118 M-1||7, 8, 9, 10|
|MEQ||315.15||L||H2O||344||3900||442||H2O||145 M-1||7, 8, 9, 10, 15|
|184.19||L||DMF, MeCN||419||9400||493||see Notes||17|
|rrhodamine 110 (R110)||366.80||L||DMSO||499||92,000||521||MeOH|
For Research Use Only. Not for use in diagnostic procedures.