Indicators for Na+, K+, Cl– and Miscellaneous Ions

CHAPTER 21

Indicators for Na+, K+, Cl and Miscellaneous Ions

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CHAPTER 21

Indicators for Na+, K+, Cl and Miscellaneous Ions

21.1 Fluorescent Na+ and K+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905SBFI and PBFI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905

Properties of SBFI and PBFI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905

Cell Loading with SBFI and PBFI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 906

Applications of SBFI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 906

Applications of PBFI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 906

Sodium Green Na+ Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 907

CoroNa Na+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908

CoroNa Green Na+ Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908

CoroNa Red Na+ Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908

FluxOR Potassium Ion Channel Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909

Assaying K+ Channels with the FluxOR Potassium Ion Channel Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 909

Using BacMam Technology for Transient Expression of K+ Channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 910

Data Table 21.1 Fluorescent Na+ and K+ Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911

Product List 21.1 Fluorescent Na+ and K+ Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 912

21.2 Detecting Chloride, Phosphate, Nitrite and Other Anions . . . . . . . . . . . . . . . . . . . . . . . . . . . 913Fluorescent Chloride Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913

SPQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

MQAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

MEQ and Cell-Permeant Dihydro-MEQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

Lucigenin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

Alternative Detection Techniques for Halides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 914

Premo Halide Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 915

Cyanide Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

Nitrite, Nitrate and Nitric Oxide Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

Measure-iT High-Sensitivity Nitrite Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916

Griess Reagent Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917

DAF-FM Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 917

2,3-Diaminonaphthalene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918

NBD Methylhydrazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918

Other Nitrate Detection Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918

Phosphate and Pyrophosphate Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919

PiPer Phosphate Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919

PiPer Pyrophosphate Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 919

EnzChek Phosphate Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 920

EnzChek Pyrophosphate Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 920

Data Table 21.2 Detecting Chloride, Phosphate, Nitrite and Other Anions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 921

Product List 21.2 Detecting Chloride, Phosphate, Nitrite and Other Anions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 922

The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies

IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

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Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions

Alexa Fluor 568 phalloidin, Oregon Green wheat germ agglutinin and Hoechst 33342.


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes




Section 21.1 Fluorescent Na+ and K+ Indicators

21.1 Fluorescent Na+ and K+ IndicatorsSodium and potassium channels are ion-selective protein pores that span the cells plasma

membrane and serve to establish and regulate membrane potential. ey are typically classi-ed according to their response mechanism: voltage-gated channels open or close in response to changes in membrane potential,1 whereas ligand-gated or ion-activated channels are triggered by ligand or ion binding.2 In excitable cells such as neurons and myocytes, these channels function both to create the action potential and to reset the cells resting membrane potential.

In this section, we describe sodium- and potassium-selective uorescent indicators, as well as the FluxOR allium Detection Kits, which provide a uorescence-based method for assaying potassium ion channel and transporter activities. e next section describes uorescent indicators for intracellular and extracellular chloride, together with an assortment of analytical reagents and methods for direct or indirect quantitation of other inorganic anions.

SBFI and PBFIProperties of SBFI and PBFI

SBFI 3 and PBFI 3,4 are uorescent indicators for sodium and potassium, respective-ly. Although the selectivity of SBFI and PBFI for their target ions is less than that of calcium indicators such as fura-2, it is sucient for the detection of physiological concentrations of Na+ and K+ in the presence of other monovalent cations.3 Furthermore, the spectral responses of SBFI and PBFI upon ion binding permit excitation ratio measurements (Loading and Calibration of Intracellular Ion IndicatorsNote 19.1), and these indicators can be used with the same optical lters and equipment used for fura-2.5,6

SBFI (Figure 21.1.1) and PBFI (Figure 21.1.2) comprise benzofuranyl uorophores linked to a crown ether chelator. e cavity size of the crown ether confers selectivity for Na+ versus K+ (or vice versa in the case of PBFI). When an ion binds to SBFI or PBFI, the indicators uorescence quantum yield increases, its excitation peak narrows and its excitation maximum shis to short-er wavelengths (Figure 21.1.3), causing a signicant change in the ratio of uorescence intensities excited at 340/380 nm (Figure 21.1.4, Figure 21.1.5). is uorescence signal is slightly sensitive to changes in pH between 6.5 and 7.5,7,8 but it is strongly aected by ionic strength 9 and viscosity.10 Researchers have described the use of SBFI for emission ratio detection 11 (410/590 nm, excited at

Figure 21.1.1 SBFI, tetraammonium salt (S1262).

Figure 21.1.2 PBFI, tetraammonium salt (P1265MP).

Figure 21.1.3 Fluorescence excitation (detected at 505 nm) and emission (excited at 340 nm) spectra of SBFI in pH 7.0 buer containing 135 mM (A) or 0 mM (B) Na+.

Figure 21.1.5 The excitation spectral response of PBFI (P1265MP) to K+: A) in Na+-free solution and B) in solutions containing Na+ with the combined K+ and Na+ concentra-tion equal to 135 mM. The scale on the vertical axis is the same for both panels.

135 mM K+

135 mM K+

Em = 505 nm

[Na+] = 0

Em = 505 nm

[K+] + [Na+] = 135 mM

Fluo

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exc

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Wavelength (nm)300 325 350 375 400

50

10881

5441

29209.9

0

302012

8.06.04.0

2.0

1.0

0

A

B

Figure 21.1.4 The excitation spectral response of SBFI (S1262) to Na+: A) in K+-free solution and B) in solutions containing K+ with the combined Na+ and K+ concentration equal to 135 mM. The scale on the vertical axis is the same for both panels.

325 350 375

135 mM Na+

135 mM Na+

Em = 505 nm[K+] = 0 mM

Em = 505 nm[Na+] + [K+] = 135 mM

Fluo

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exc

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Wavelength (nm)300 400

30

9045

3016

9.66.74.0

2.0

1.00

169.66.7

3.22.31.6

0.8

0

A

B



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340 nm). More recently, the implementation of two-photon excitation of SBFI with infrared light has been reported for Na+ imaging in spines and ne dendrites of central neurons 12,13 (Figure 21.1.6).

Although SBFI is quite selective for the Na+ ion, K+ has some ef-fect on the native anity of SBFI for Na+ (Figure 21.1.4). e dissocia-tion constant (Kd) of SBFI for Na+ is 3.8 mM in the absence of K+, and 11.3 mM in solutions with a combined Na+ and K+ concentration of 135 mM, which approximates physiological ionic strength. SBFI is ~18-fold more selective for Na+ than for K+. Likewise, the Kd of PBFI for K+ is strongly dependent on whether Na+ is present (Figure 21.1.5), with a value of 5.1 mM in the absence of Na+ and 44 mM in solutions with a combined Na+ and K+ concentration of 135 mM. In buers in which the Na+ is replaced by tetramethylammonium chloride, the Kd of PBFI for K+ is 11 mM; choline chloride and N-methylglucamine are two other possible replacements for Na+ in the medium. Although PBFI is only 1.5-fold more selective for K+ than for Na+, this selectivity is oen suf-cient because intracellular K+ concentrations are normally about 10 times higher than Na+ concentrations.

e Kd of all ion indicators depends on factors such as pH, tem-perature, ionic strength, concentrations of other ions and dyeprotein interactions. Due to these environmental factors, the Kd determined in situ for intracellular SBFI is substantially higher than that determined in cell-free buer solutions. Kd (Na+) values of 29 mM, 26.6 mM and 18.0 mM have been determined for SBFI in lizard peripheral axons, porcine adrenal chroman cells and rat hippocampal neurons, re-spectively.7,14 Consequently, intracellular SBFI should be calibrated using the pore-forming antibiotic gramicidin 5 (G6888). Palytoxin, an ionophoric toxin isolated from marine coelenterates, is much more ef-fective than gramicidin for equilibrating intracellular and extracellular Na+.14 Intracellular PBFI should be calibrated using the K+ ionophore valinomycin 15 (V1644).

Figure 21.1.6 CA1 pyramidal neuron in a hippocampal slice lled with SBFI (S1262) deliv-ered from a patch pipette (visible on the right). The image was obtained using two-photon excitation of SBFI at 790 nm. Image contributed by Christine R. Rose, Physiological Institute, University of Munich.

Cell Loading with SBFI and PBFISBFI and PBFI are available both as cell-impermeant acid salts (S1262,

P1265MP) and as cell-permeant acetoxymethyl (AM) esters (S1263, S1264, P1267MP). e anionic acid forms can be loaded into cells using our Inux pinocytic cell-loading reagent (I14402, Section 19.8), or by micro-injection, patch-pipette infusion or electroporation. For AM ester loading (Loading and Calibration of Intracellular Ion IndicatorsNote 19.1), ad-dition of the Pluronic F-127 (P3000MP, P6866, P6867) or PowerLoad (P10020) dispersing agents as well as relatively long incubation timesup to four hoursare typically necessary.5 ATP-induced permeabilization reportedly produces increased uptake of SBFI AM by bovine pulmonary arterial endothelial cells 16 (BPAEC). Somewhat higher working concen-trations of PBFI and SBFI than those used for fura-2 may be required be-cause of the lower uorescence quantum yields of these indicators. AM ester loading sometimes produces intracellular compartmentalization of SBFI.10,17 As with other AM esters, reducing the incubation temperature below 37C may inhibit compartmentalization. Other practical aspects of loading and calibrating SBFI have been reviewed by Negulescu and Machen.5

Applications of SBFISBFI has been employed to estimate Na+ gradients in isolated mito-

chondria,1719 as well as to measure intracellular Na+ levels or Na+ eux in cells from a variety of tissues:

Bloodplatelets,20 monocytes 21 and lymphocytes 22

Brainastrocytes,23 neurons,12,24 and presynaptic terminals 25,26

Muscleperfused heart,27,28 cardiomyocytes 2932 and smooth muscle 33,34

Secretory epithelia 3537

Plants 38

SBFI has also been used in combination with other uorescent in-dicators to correlate changes in intracellular Na+ with Ca2+ and Mg2+ concentrations,24,39,40 intracellular pH and membrane potential.21

Applications of PBFIPBFI 4 has fewer documented applications than SBFI. Renewed in-

terest has been prompted by the observation that intracellular K+ levels appear to be a controlling factor in apoptotic cell death pathways.41 Flow cytometric measurements using UV argon-ion laser excitation (351 nm and 364 nm) of PBFI indicate that K+ eux induces shrinkage of apop-totic cells and is a trigger for caspase activation.4245 Furthermore, PBFI provides a potential alternative to radiometric 86Rb eux assays for quantitating K+ transport.15 Other applications of PBFI include:

Detecting adrenoceptor-stimulated decreases of intracellular K+ concentration in astrocytes and neurons 46

Evaluating the mediating eects of K+ depletion on monocytic cell necrosis 47

Investigating the relationship between cytoplasmic K+ concentra-tions and NMDA excitotoxicity 48

Measuring intracellular K+ uxes associated with apoptotic cell shrinkage 49,50

Monitoring mitochondrial KATP channel activation 5153

Quantitating K+ in isolated cochlear outer hair cells 54 and in mam-malian ventricles using patch-clamp techniques 55







Detecting elevated intracellular K+ levels associated with HIV-induced cytopathology 56

Measuring K+ levels in plant cells and vacuoles 57

Sodium Green Na+ Indicatore Sodium Green indicator can be excited at 488 nm (Figure

21.1.7), providing a valuable alternative to the UV lightexcitable SBFI for use with confocal laser-scanning microscopes 58 and ow cytom-eters.59 We oer the cell-impermeant tetra(tetramethylammonium) salt of the Sodium Green indicator (S6900), as well as its cell-permeant tetraacetate (S6901).

e Sodium Green indicator comprises two 2 ,7-dichlorouo-rescein dyes linked to the nitrogen atoms of a crown ether (Figure 21.1.8) with a cavity size that confers selectivity for the Na+ ion. Upon binding Na+, the Sodium Green indicator exhibits an increase in uorescence emission intensity with little shi in wavelength (Figure 21.1.9). Although the Sodium Green indicator lacks the direct ratio-metric readout capability of SBFI, uorescence intensity uctuations due to cell size variability can be compensated to some extent by using forward light scatter as a reference signal in ow cytometry.59

As compared with SBFI, the Sodium Green indicator shows greater selectivity for Na+ than K+ (~41-fold versus ~18-fold) and dis-plays a much higher uorescence quantum yield (0.2 versus 0.08) in Na+-containing solutions. e longer-wavelength absorption of the Sodium Green indicator results in reduction of the potential for

Figure 21.1.8 Sodium Green, tetra (tetramethylammo-nium) salt (S6900).

Figure 21.1.9 Emission spectral response of the Sodium Green indicator (S6900) to Na+: A) in K+-free solution and B) in solutions containing K+ with the combined Na+ and K+ concentration equal to 135 mM. The scale on the vertical axis is the same for both panels.

Ex = 488 nm[K+] = 0 mM

13107

4

2

1

0

A

B

Wavelength (nm)

Fluo

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ence

em

issi

on

475 525 575 625

135 mM Na+

135 mM Na+

9473

3726

14

7

0

Ex = 488 nm[Na+] + [K+] = 135 mM

Figure 21.1.7 Absorption and uorescence emission spec-tra of Sodium Green indicator in pH 7.0 buer containing 135 mM Na+.

photodamage to the cell because the energy of the excitation light is lower than that of the UV light required for excitation of SBFI. e Kd of the Sodium Green indicator for Na+ is about 6 mM in K+-free solution and about 21 mM in solutions with combined Na+ and K+ concentration of 135 mM, approximating physiological ionic strength. Because its Kd may be shied due to intracellular interac-tions, the Sodium Green indicator should be calibrated in situ us-ing the pore-forming antibiotic gramicidin 59 (G6888). In some cases, dyeprotein interactions may cause severe dampening or even com-plete elimination of the Na+-dependent uorescence response of in-tracellular Sodium Green indicator. Nevertheless, ow cytometric measurements in Chinese hamster ovary (CHO) cells are well cor-related with spectrouorometric measurements using SBFI.59 Other applications include:

Assessing the regulation of Na+/K+-ATPase by persistent Na+ accumulation in rat thalamic neurons 60

Confocal imaging of Na+ transport in rat colonic mucosa 61 and cochlear hair cells by ow cytometry 62

Detecting anoxia-induced Na+ inux in neurons 63

Determining intracellular Na+ concentration in craysh presynap-tic terminals using an area-ratio method 64

Fluorescence lifetime imaging of intracellular Na+ 6567

Measuring intracellular Na+ concentration in bacterial cells 68 and green algae 69

Determining voltage-gated sodium channel NaV1.5driven endosomal Na+ levels in macrophages 21








CoroNa Na+ IndicatorsCoroNa Green Na+ Indicator

e CoroNa Green dye is a green-uorescent Na+ indicator that exhibits an increase in uorescence emission intensity upon binding Na+ (excitation/emission = 492/516 nm), with little shi in wavelength (Figure 21.1.10). Similar to our SBFI and Sodium Green Na+ indicators, the CoroNa Green indicator allows spatial and temporal resolution of Na+ concentrations in the presence of physiological concentrations of other monovalent cations.7073 CoroNa Green Na+ indicator has been co-loaded with Alexa Fluor 594 dextran (an ion-insensitive reference) via suction pipettes into live rat optic nerves for confocal imaging of intracellular Na+ levels; calcium measurements were also made using uo-4 dextran and Alexa Fluor 594 dextran.74

Comprising a uorescein molecule linked to a crown ether with a cavity size that confers selectivity for the Na+ ion (Figure 21.1.11), the CoroNa Green indicator is less than half the size of the Sodium Green indicator 75 (molecular weight 586 and 1668, respectively). is smaller size appears to help the cell-permeant CoroNa Green AM (Figure 21.1.12) load cells more eectively than the Sodium Green tetraacetate. Furthermore, the CoroNa Green indicator responds to a broader range of Na+ concentration, with a Kd of ~80 mM. e cell-impermeant CoroNa Green indicator (C36675) is supplied in a unit size of 1 mg. e cell-permeant AM ester of the CoroNa Green indicator (C36676) is supplied as a set of 20 vials, each containing 50 g of the indicator.

CoroNa Red Na+ IndicatorCoroNa Red chloride is based on a crown ether that has structural similarity to the Ca2+

chelator BAPTA (Figure 21.1.13). Unlike SBFI and the Sodium Green indicator, the net posi-tive charge of CoroNa Red chloride targets the indicator to mitochondria (Figure 21.1.14), and therefore loading of cells does not require use of a permeant ester derivative of the dye. Cells are typically loaded by adding 0.51.0 M CoroNa Red chloride from a 1 mM stock solution in DMSO, incubating for 1030 minutes at 37C and nally washing with dye-free medium before commencing uorescence analysis. e CoroNa Red indicator is only weakly uorescent in the absence of Na+ and its uorescence increases ~15-fold upon binding Na+ (Figure 21.1.15). Despite its relatively high Kd for Na+ of ~200 mM, the CoroNa Red indicator exhibits sensitive responses to cellular Na+ inuxes through voltage-gated channels and ATP-gated cation pores. Verkman and co-workers have immobilized the CoroNa Red indicator on polystyrene microspheres and used this complex to measure Na+ concentrations around 100 mM in the tracheal airwaysurface liquid (ASL) of cultured epithelial cells and human lung tissues.76,77 e CoroNa Red indicator has also been employed to investigate the Na+ channel permeation pathway using polyhistidine-tagged and pore-only constructs of a voltage-dependent Na+ channel.78 e CoroNa Red indica-tor is available as a single 1 mg vial (C24430) or as a set of 20 vials, each containing 50 g of the indicator (C24431).

Figure 21.1.11 CoroNa Green (C36675).

O

N

O

O

O

O OHO

FF

CH3OCCH2O

Figure 21.1.14 Images of an NIH 3T3 cell showing colocalization of the CoroNa Red sodium indicator (left panel; C24430, C24431) with the MitoTracker Green FM mitochondrial marker (right panel, M7514). A cell loaded with both dyes was imaged consecutively using Omega Optical bandpass lter set XF41 for CoroNa Red sodium indicator and set XF23 for MitoTracker Green FM.

Figure 21.1.13 CoroNa Red chloride (C24430).

Figure 21.1.10 Fluorescence emission spectra of the CoroNa Green indicator (C36675, C36676) in 50 mM MOPS, pH 7.0 (adjusted with tetramethylammonium hydroxide), containing 100 mM K+ and variable concentrations of Na+ as indicated.

Wavelength (nm)

500 510 530 550

Ex = 485 nm

Fluo

resc

ence

em

issi

on

520 540

100 mM

250 mM

1000 mM

50 mM

0 mM

Figure 21.1.12 CoroNa Green, AM (C36676).

O

N

O

O

O

O OCH3COCH2O

FF

CH3OCCH2O

O







FluxOR Potassium Ion Channel AssayAssaying K+ Channels with the FluxOR Potassium Ion Channel Assay Kit

e FluxOR Potassium Ion Channel Assay Kits (F10016, F10017) provide a uorescence-based assay for high-throughput screening (HTS) of potassium ion channel and transporter activities.7981 e FluxOR Potassium Ion Channel Assay Kits take advantage of the well-de-scribed permeability of potassium channels to thallium (Tl+) ions. When thallium is present in the extracellular solution containing a stimulus to open potassium channels, channel activity is detected with a cell-permeant thallium indicator dye that reports large increases in uorescence emission at 525 nm as thallium ows down its concentration gradient and into the cells (Figure 21.1.16). In this way, the uorescence reported in the FluxOR system becomes a surrogate in-dicator of activity for any ion channel or transporter that is permeable to thallium, including the human ether-a-go-gorelated gene (hERG) channel, one of the human cardiac potassium channels. e FluxOR potassium ion channel assay has been validated for homogeneous high-throughput proling of hERG channel inhibition using BacMam-mediated transient expression of hERG.80 e FluxOR Potassium Ion Channel Assay Kits can also be used to study potassium co-transport processes that accommodate the transport of thallium into cells.82 Furthermore, resting potassium channels and inward rectier potassium channels like Kir2.1 can be assayed by adding stimulus buer with thallium alone, without any depolarization to measure the signal.

e FluxOR reagent, a thallium indicator dye, is loaded into cells as a membrane-permeable AM ester. e FluxOR dye is dissolved in DMSO and further diluted with FluxOR assay buer, a physiological HBSS (Hanks balanced salt solution), for loading into cells. Loading is assisted by the proprietary PowerLoad concentrate, an optimized formulation of nonionic Pluronic surfactant polyols that act to disperse and stabilize AM ester dyes for optimal loading in aqueous solution. is PowerLoad concentrate is also available separately (P10020) to aid the solubiliza-tion of water-insoluble dyes and other materials in physiological media.

Once inside the cell, the nonuorescent AM ester of the FluxOR dye is cleaved by endog-enous esterases into a weakly uorescent (basal uorescence), thallium-sensitive indicator. e thallium-sensitive form is retained in the cytosol, and its extrusion is inhibited by water-soluble probenecid (P36400, Section 19.8), which blocks organic anion pumps. For most applications, cells are loaded with the dye at room temperature. For best results, the dye-loading buer is then replaced with fresh, dye-free assay buer (composed of physiological HBSS containing proben-ecid), and cells are ready for the HTS assay.

Each FluxOR Potassium Ion Channel Assay Kit contains:

Figure 21.1.15 Fluorescence emission spectra of the CoroNa Red indicator (C24430, C24431) in 50 mM MOPS (pH 7.0, adjusted with tetramethylammonium hydroxide) containing 100 mM K+ and variable concentrations of Na+ as indicated.

550 575

Wavelength (nm)

Fluo

resc

ence

em

issi

on

600 625 650

Ex = 545 nm2000 mM Na+

1000

500

250

100

50

250

Figure 21.1.16 Thallium redistribution in the FluxOR assay. Basal uorescence from cells loaded with FluxOR reagent (pro-vided in the FluxOR Potassium Ion Channel Assay Kits; F10016, F10017) is low when potassium channels remain unstimu-lated, as shown in the left panel. When thallium is added to the assay with the stimulus, the thallium ows down its concentra-tion gradient into the cells, activating the dye as shown in the right panel.

Ion channel Ion channel

Tl +

Tl+ Tl +

Tl+

Tl+

Tl+

Tl+

Tl+

Tl +

Closed

Open Tl +

Tl+

Tl+

Tl +

Tl+

StimulatedResting

Thallium Dye

Extracellular Intracellular Extracellular Intracellular

Tl +

Tl+

Tl+

Tl+

FluxOR reagent FluxOR assay buer PowerLoad concentrate Probenecid FluxOR chloride-free buer

Potassium sulfate (K2SO4) concentrate allium sulfate (Tl2SO4) concentrate Dimethylsulfoxide (DMSO) Detailed protocols



thermofi sher.com/probes





e FluxOR Kits provide a concentrated thallium solution along with sucient dye and buers to perform ~4000 (F10016) or ~40,000 (F10017) assays in a 384-well microplate format. ese kits allow max-imum target exibility and ease of operation in a homogeneous for-mat. e FluxOR potassium ion channel assay has been demonstrated for use with CHO and HEK 293 cells stably expressing hERG, as well as U2OS cells transiently transduced with the BacMam hERG reagent 80 (B10019, B10033; see below) (Figure 21.1.17). More information is avail-able at www.invitrogen.com/handbook/uxorpotassium.

Using BacMam Technology for Transient Expression of K+ Channels

Potassium channel cDNAs that have been engineered into a bacu-lovirus gene delivery/expression system using BacMam technology (BacMam Gene Delivery and Expression TechnologyNote 11.1) are also available for use with the FluxOR Potassium Ion Channel Assay Kits, including the human ether-a-go-go related gene 80 (hERG) (Figure 21.1.18), several members of the voltage-gated K+ channel (Kv) gene family and two members of the inwardly rectifying K+ channel (Kir) gene family:

BacMam hERG (for 10 microplates, B10019; for 100 microplates, B10033)

BacMam Kv1.1 (for 10 microplates, B10331) BacMam Kv1.3 (for 10 microplates, B10332) BacMam Kv2.1 (for 10 microplates, B10333) BacMam Kv7.2 and Kv7.3 (for 10 microplates, B10147) BacMam Kir1.1 (for 10 microplates, B10334) BacMam Kir2.1 (for 10 microplates, B10146)

e BacMam system uses a modied insect cell baculovirus as a vehicle to eciently deliver and express genes in mammalian cells with minimum eort and toxicity. e use of BacMam delivery in mammalian cells is relatively new, but well described, and has been used extensively in a drug discovery setting.83 Constitutively ex-pressed ion channels and other cell surface proteins have been shown to contribute to cell toxicity in some systems, and may be subject to clonal dri and other inconsistencies that hamper successful experi-mentation and screening. us, inducible, division-arrested or tran-sient expression systems such as BacMam technology are increasingly methods of choice to decrease variability of expression in such assays.

U2OS cells (ATCC number HTB-96) have been shown to dem-onstrate highly ecient expression of BacMam-delivered targets in a null background ideal for screening in a heterologous expression sys-tem. e U2OS cell line is recommended for use if your particular cell line does not eciently express the BacMam targets. Examples of other cell lines that are eciently transduced by BacMam technology include HEK 293, HepG2, BHK, Cos-7 and Saos-2.

Figure 21.1.18 BacMam-hERG gene delivery and expression. This schematic depicts the mechanism of BacMam-mediated gene delivery into a mammalian cell and expression of the hERG gene (B10019, B10033). The hERG gene resides within the baculoviral DNA, down-stream of a CMV promoter that drives its expression when introduced into a mammalian target cell. BacMam viral particles are taken up by endocytic pathways into the cell, and the DNA within them is released for transcription and expression. The translated protein is then folded for insertion into the membrane, forming functional hERG ion channels. This process begins within 46 hours and in many cell types is completed after an overnight period.

Promoter human Ether--go-go Related Gene

Baculovirus

hERG Gene

Endocytotic entry

DNA movesto nucleus hERG gene

transcribed

DNA

mRNA

mRNAtranslated

Assembly

Ion channel

Membraneinsertion

Assembly

Figure 21.1.17 FluxOR potassium ion channel assays (F10016, F10017) performed on fresh and frozen U2OS cells transduced with the BacMam hERG reagent (B10019, B10033). A) Raw data (RFU = relative uorescence units) obtained in the FluxOR assay determination of thal-lium ux in U2OS cells, which had been transduced with BacMam-hERG and kept frozen until the day of use. The arrow indicates the addition of the thallium/potassium stimulus, and up-per and lower traces indicate data taken from the minimum and maximum doses of cisapride used in the determination of the dose-response curves. B) Raw pre-stimulus peak and base-line values were boxcar averaged and normalized to indicate the fold increase in uorescence over time. C) Data generated in a dose-response determination of cisapride block on BacMam hERG expressed in U2OS cells freshly prepared from overnight expression after viral transduc-tion. D) Parallel data obtained from cells transduced with BacMam-hERG, stored for 2 weeks in liquid nitrogen, thawed and plated 4 hours prior to running the assay. Error bars indicate standard deviation, n = 4 per determination.

39

34

29

24

19

14

9

Time (sec)

0 604020 80 100 120

103

RFU

Cisapride block orBacMam negative control

U-2 OSBacMam hERG

A

2.0

1.8

1.4

1.6

1.2

1.0

0.8

0.6

0.4

0.2

F

/F

[Cisapride] (nM)

IC50 = 73 nM

Fresh

10-1 102101100 103 104 105

C

2.5

2.3

2.1

1.9

1.7

1.5

1.3

1.1

0.9

F

/F

Time (sec)

0 604020 80 100 120

Cisapride block orBacMam negative control

U-2 OSBacMam hERG

B

2.0

1.8

1.4

1.6

1.2

1.0

0.8

0.6

0.4

0.2

F

/F

[Cisapride] (nM)

IC50 = 79 nM

Frozen

10-1 102101100 103 104 105

D


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofi sher.com/probes





DATA TABLE 21.1 FLUORESCENT Na+ AND K+ INDICATORS Low Ion* High Ion*

Cat. No. MW Storage Soluble Abs EC Em Solvent Abs EC Em Solvent Product Kd NotesC24430 773.32 L DMSO 547 92,000 570 H2O 551 92,000 576 H2O/Na+ 200 mM 1, 2, 3, 4C24431 773.32 L DMSO 547 92,000 570 H2O 551 92,000 576 H2O/Na+ 200 mM 1, 2, 3, 4C36675 585.56 F,D,L pH >6 492 68,000 516 H2O 492 68,000 516 H2O/Na+ 80 mM 1, 2, 5, 6C36676 657.62 F,D,L DMSO 454 23,000 516 pH 7 C36675G6888 ~1880 D MeOH 6 336 33,000 557 H2O 338 41,000 507 H2O/K+ 5.1 mM 1, 5, 7P1267MP 1171.13 F,D,L DMSO 369 37,000 see Notes MeOH P1265MP 8S1262 906.94 L pH >8 339 45,000 565 H2O 333 52,000 539 H2O/Na+ 3.8 mM 1, 5, 9S1263 1127.07 F,D,L DMSO 379 32,000 see Notes MeOH S1262 8S1264 1127.07 F,D,L DMSO 379 32,000 see Notes MeOH S1262 8S6900 1667.57 L pH >6 506 117,000 532 H2O 507 133,000 532 H2O/Na+ 6.0 mM 1, 2, 5, 9S6901 1543.17 F,D,L DMSO 302 21,000 none MeOH S6900V1644 1111.33 F,L EtOH 10-fold excess of free cation X (H2O/X) relative to the listed dissociation constant (Kd) for cation X.6. Kd(Na+) determined in 50 mM MOPS, pH 7.0 (adjusted with tetramethylammonium hydroxide) at 22C.7. Kd(K+) has been determined in 10 mM MOPS, pH 7.0 (adjusted with tetramethylammonium hydroxide) at 22C. Kd(K+) is strongly dependent on the concentration of Na+.

In solutions with Na+ + K+ = 135 mM, Kd(K+) = 44 mM.8. Fluorescence of SBFI AM and PBFI AM is very weak.9. Kd(Na+) has been determined in 10 mM MOPS, pH 7.0 (adjusted with tetramethylammonium hydroxide) at 22C. Na+ dissociation constants for these indicators are dependent on K+

concentration. In solutions with total Na+ + K+ = 135 mM, Kd(Na+) = 11.3 mM (S1262) and 21 mM (S6900).

1. Nat Rev Drug Discov (2009) 8:982; 2. Neuropharmacology (2009) 56:2; 3. J Biol Chem (1989) 264:19449; 4. Biophys J (1995) 68:2469; 5. Methods Enzymol (1990) 192:38; 6. Proc Natl Acad Sci U S A (1997) 94:7053; 7. Am J Physiol Cell Physiol (2001) 280:C1623; 8. J Physiol (1997) 498:295; 9. Cell Regul (1990) 1:259; 10. J Biol Chem (1989) 264:19458; 11. J Mol Cell Cardiol (1997) 29:3375; 12. J Neurosci (2001) 21:4207; 13. Pugers Arch (1999) 439:201; 14. J Neurosci Methods (1997) 75:21; 15. J Biol Chem (1990) 265:10522; 16. J Appl Physiol (1996) 81:509; 17. J Physiol (1992) 448:493; 18. J Biol Chem (1995) 270:672; 19. Am J Physiol (1992) 262:C1047; 20. J Biol Chem (2004) 279:19421; 21. J Immunol (2007) 178:7822; 22. J Biol Chem (2006) 281:2232; 23. J Physiol (2009) 587:5859; 24. J Neurosci (2009) 29:7803; 25. Biophys J (1997) 73:2476; 26. J Neurochem (1998) 70:1513; 27. Am J Physiol Heart Circ Physiol (2001) 280:H280; 28. Am J Physiol (1997) 273:H1246; 29. Am J Physiol Heart Circ Physiol (2000) 279:H1661; 30. Am J Physiol Heart Circ Physiol (2000) 279:H2143; 31. J Mol Cell Cardiol (1997) 29:2653; 32. Am J Physiol (1996) 270:H2149; 33. Biophys J (1997) 73:3371; 34. Proc Natl Acad Sci U S A (1993) 90:8058; 35. J Biol Chem (2008) 283:4602; 36. Am J Physiol Cell Physiol (2000) 279:C1648; 37. Am J Physiol Gastrointest Liver Physiol (2000) 278:G400; 38. J Exp Bot (2005) 56:3149; 39. J Pharmacol Exp er (2002) 300:9; 40. J Biol Chem (2001) 276:13657; 41. Curr Opin Cell Biol (2001) 13:405;

42. J Biol Chem (2000) 275:19609; 43. J Biol Chem (1999) 274:21953; 44. J Biol Chem (1997) 272:32436; 45. J Biol Chem (1997) 272:30567; 46. Neurosci Lett (1997) 238:33; 47. Am J Physiol (1999) 276:C717; 48. Mol Pharmacol (1999) 56:619; 49. J Biol Chem (2008) 283:36071; 50. J Biol Chem (2008) 283:7219; 51. J Biol Chem (2004) 279:32562; 52. J Biol Chem (1998) 273:13578; 53. J Biol Chem (1996) 271:8796; 54. Brain Res (1994) 636:153; 55. Circ Res (1994) 74:829; 56. J Virol (1996) 70:5447; 57. J Exp Bot (2003) 54:2035; 58. Methods Enzymol (1999) 307:119; 59. Cytometry (1995) 21:248; 60. J Physiol (2000) 525:343; 61. J Physiol (1999) 514:211; 62. Hear Res (1998) 119:1; 63. Brain Res (1994) 663:329; 64. J Neurosci Methods (2002) 118:163; 65. Anal Biochem (2000) 281:159; 66. Anal Biochem (1997) 250:131; 67. Methods Enzymol (1994) 240:723; 68. Biophys J (2006) 90:357; 69. J Biol Chem (2008) 283:15122; 70. Neuron (2009) 61:259; 71. J Neurochem (2009) 108:126; 72. J Biol Chem (2008) 283:9377; 73. J Neurosci Methods (2006) 155:251; 74. J Neurosci (2009) 29:1796; 75. Bioorg Med Chem Lett (2005) 15:1851; 76. Proc Natl Acad Sci U S A (2001) 98:8119; 77. J Clin Invest (2001) 107:317; 78. J Biol Chem (2002) 277:24653; 79. J Biomol Screen (2010) 15:441; 80. Anal Biochem (2009) 394:30; 81. Assay Drug Dev Technol (2008) 6:765; 82. J Biol Chem (2009) 284:14020; 83. Drug Discov Today (2007) 12:396.

REFERENCES








PRODUCT LIST 21.1 FLUORESCENT Na+ AND K+ INDICATORSCat. No. Product QuantityB10019 BacMam-hERG *for 10 microplates* 1 kitB10033 BacMam-hERG *for 100 microplates* 1 kitB10334 BacMam Kir1.1 *for 10 microplates* 1 kitB10146 BacMam Kir2.1 *for 10 microplates* 1 kitB10331 BacMam Kv1.1 *for 10 microplates* 1 kitB10332 BacMam Kv1.3 *for 10 microplates* 1 kitB10333 BacMam Kv2.1 *for 10 microplates* 1 kitB10147 BacMam Kv7.2 and Kv7.3 *for 10 microplates* 1 kitC36675 CoroNa Green *cell impermeant* 1 mgC36676 CoroNa Green, AM *cell permeant* *special packaging* 20 x 50 gC24430 CoroNa Red chloride 1 mgC24431 CoroNa Red chloride *special packaging* 20 x 50 gF10016 FluxOR Potassium Ion Channel Assay *for 10 microplates* 1 kitF10017 FluxOR Potassium Ion Channel Assay *for 100 microplates* 1 kitG6888 gramicidin 100 mgP1267MP PBFI, AM *cell permeant* *special packaging* 20 x 50 gP1265MP PBFI, tetraammonium salt *cell impermeant* 1 mgP6866 Pluronic F-127 *10% solution in water* *0.2 m ltered* 30 mLP3000MP Pluronic F-127 *20% solution in DMSO* 1 mLP6867 Pluronic F-127 *low UV absorbance* 2 gP10020 PowerLoad concentrate, 100X 5 mLS1263 SBFI, AM *cell permeant* 1 mgS1264 SBFI, AM *cell permeant* *special packaging* 20 x 50 gS1262 SBFI, tetraammonium salt *cell impermeant* 1 mgS6901 Sodium Green tetraacetate *cell permeant* *special packaging* 20 x 50 gS6900 Sodium Green, tetra(tetramethylammonium) salt *cell impermeant* 1 mgV1644 valinomycin 25 mg






Section 21.2 Detecting Chloride, Phosphate, Nitrite and Other Anions

21.2 Detecting Chloride, Phosphate, Nitrite and Other Anions

is section describes uorescent indicators for intracellular and extracellular chloride to-gether with an assortment of analytical reagents and methods for direct or indirect quantita-tion of other inorganic anions, including bromide, iodide, hypochlorite, cyanide, nitrite, nitrate, phosphate, pyrophosphate and selenide.1

Fluorescent Chloride IndicatorsMost of the uorescent chloride indicators are 6-methoxyquinolinium derivatives, the proto-

type of which is 6-methoxy-N-(3-sulfopropyl)quinolinium 2,3 (SPQ, Figure 21.2.1). Cl detection sensitivity has been improved by modications of the quinolinium N substituent.4,5 Our current range of Cl indicators consists of:

6-Methoxy-N-(3-sulfopropyl)quinolinium (SPQ, M440) N-(Ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE, E3101) 6-Methoxy-N-ethylquinolinium iodide (MEQ, M6886) Lucigenin (L6868)

All of these indicators detect Cl via diusion-limited collisional quenching.6 is detection mechanism is dierent from that of uorescent indicators for Ca2+, Mg2+, Zn2+, Na+ and K+. It involves a transient interaction between the excited state of the uorophore and a halide ionno ground-state complex is formed. Quenching is not accompanied by spectral shis (Figure 21.2.2) 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 ecient than Cl quenching.6 Fortunately, physiological concentrations of non-choloride ions do not signicantly aect the uorescence of SPQ and other methoxyquinolinium-based Cl indicators. With some excep-tions,7 uorescence of these indicators is not pH sensitive in the physiological range.4 Because Cl-dependent uorescence quenching is a diusional process, it is quite sensitive to solution viscosity and volume. Exploiting this property, SPQ has been used to measure intracellular vol-ume changes.8

e eciency of collisional quenching is characterized by the SternVolmer constant (KSV), dened as the reciprocal of the ion concentration that produces 50% of maximum quenching. For SPQ, KSV is reported to be 118 M1 in aqueous solution and 12 M1 inside cells.9 For MQAE, in situ KSV values of 2528 M1 have been determined in various cell types,10,11 compared with the solution value of 200 M1. Intracellular Cl indicators are generally calibrated using high-K+ buers and the K+/H+ ionophore nigericin (N1495) in conjunction with tributyltin chloride, an organometallic compound that acts as a Cl/OH antiporter.4,12 With the exception of diH-MEQ, 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.1012

Measurement of intracellular Cl concentrations and the study of Cl channels have been stimulated by the discovery that cystic brosis is caused by mutations in a gene encoding a Cl transport channel, which is known as the cystic brosis transmembrane conductance regula-tor 13 (CFTR). Cl permeability assays are used to detect activity of the CFTR and other anion transporters.1417 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 re-placed by nitrate (NO3). NO3 is used in this assay protocol because it produces no uorescence quenching of the indicator, yet its channel permeability is essentially the same as that of Cl14,15 (Figure 21.2.3).

Figure 21.2.1 6-methoxy-N-(3-sulfopropyl)quinolinium, inner salt (SPQ, M440).

Figure 21.2.2 Fluorescence emission spectra of MQAE (E3101) in increasing concentrations of Cl.

0 mM CI-

1.02.0

3.05.0

7.012

25100

Ex = 350 nm

Fluo

resc

ence

em

issi

on

Wavelength (nm)400 450 500 550 600

Figure 21.2.3 Detection of cystic brosis transmem-brane 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 = uo-rescence intensity in absence of chloride, F = uorescence intensity at time points indicated on the x-axis). Upon ad-dition of cyclic AMP to initiate channel opening, and ex-change of extracellular Cl (135 mM) for nitrate (NO3), SPQ quenching decreases in CFTR-expressing cells (lled 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.

1.0

1.5

2.0

2.5

Cl- Cl-NO3-cAMP

F 0 /F

2 4 6 8 10

Time (minutes)








SPQSPQ (M440, Figure 21.2.1) is currently in widespread use for detect-

ing CFTR activity using the Cl/NO3 exchange technique described above.16,1824 SPQ has also has been employed to investigate Cl uxes through several other transporters such as the GABAA receptor,25,26 erythrocyte Cl/HCO3 exchangers 27,28 and the mitochondrial uncou-pling protein.2931 Although SPQ requires UV excitation (as do MQAE and MEQ), techniques for ow cytometric detection and calibration of the indicator using argon-ion laser excitation at 351 nm and 364 nm have been successfully demonstrated.12

MQAEMQAE (E3101, Figure 21.2.4) has greater sensitivity to Cl4,5 and a

higher uorescence quantum yield than SPQ; consequently, it is cur-rently 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 uorescence response.32 MQAE has been used in a uorescence-based microplate assay that has potential for screening compounds that modify Cl ion-channel activity.10 Other applications have included Cl measurements in cytomegalovirus-infected broblasts,33 smooth mus-cle cells 32 and salivary glands,17 as well as in reconstituted membranes containing the GABAA receptor 26 or the mitochondrial-uncoupling protein 34,35 (UCP-1).

MEQ and Cell-Permeant Dihydro-MEQe Cl indicator 6-methoxy-N-ethylquinolinium iodide (MEQ)

can be rendered cell-permeant by masking its positively charged ni-trogen to create a lipophilic, Cl-insensitive compound, 6-methoxy-N-ethyl-1,2-dihydroquinoline 36 (dihydro-MEQ). is reduced quino-line derivative can then be loaded noninvasively into cells, where it is rapidly reoxidized in most cells to the cell-impermeant, Cl-sensitive MEQ (Figure 21.2.5). Using this technique, researchers have loaded live brain slices and hippocampal neurons with MEQ for confocal imag-ing of Cl responses to GABAA receptor activation and glutamatergic

Figure 21.2.5 Intracellular delivery of the uorescent chloride indicator 6-methoxy-N-ethylquino-linium iodide (MEQ, M6886), via oxidation of the membrane-permeant precursor dihydro-MEQ.

Dihydro-MEQ MEQ

Cell membrane

OxidationNCH

2CH

3

CH3

O

N

CH2

CH3

CH3

O

+

Figure 21.2.6 Lucigenin (bis-N-methylacridinium nitrate, L6868).

excitotoxicity.3741 Quenching of intracellular MEQ uorescence by Cl has a KSV of 19 M1, a value that is slightly higher than that reported for SPQ in broblasts. MEQ is available in solid form (M6886) and is supplied with a simple protocol for reducing it to dihydro-MEQ with sodium borohydride (not supplied) just prior to cell loading.

Lucigenine uorescence of lucigenin (L6868, Figure 21.2.6) is quantita-

tively quenched by high levels of Cl with a reported KSV = 390 M1.42 Lucigenin absorbs maximally at both 368 nm (EC = 36,000 cm1M1) and 455 nm (EC = 7400 cm1M1), with an emission maximum at 505 nm. Its uorescence emission has a quantum yield of ~0.6 and is in-sensitive to nitrate, phosphate and sulfate. Lucigenin is a useful Cl in-dicator in liposomes and reconstituted membrane vesicles; however, be-cause its uorescence is reported to be unstable in the cytoplasm, it may not always be suitable for determining intracellular Cl.42 Lucigenin has been used to detect chloride uptake in tonoplast vesicles 43 and to measure Cl inux across the pleural surface in perfused mouse lungs.44

Alternative Detection Techniques for HalidesAs mentioned above, the uorescence of SPQ and related Cl indi-

cators is quenched by collision with a variety of anions, including (in order of increasing quenching eciency) Cl, Br, I and thiocyanate 45 (SCN). For example, uorescence of SPQ is partially quenched by the anionic pH buer TES (N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid) but not by the protonated TES zwitterion, a property that has been exploited to measure proton eux from proteoliposomes.31,46 Anion detectability using diusional uorescence quenching of these uorophores is typically limited to the millimolar range. I quenches many other uorophores and is commonly used to determine the ac-cessibility of uorophores to quenching in proteins and membranes.47,48

In addition, halides can be oxidized to hypohalites (OCl, OBr, OI), which react with rhodamine 6G (R634, Section 12.2) to yield chemilu-minescent products.49,50 A cell produces OCl by oxidizing Cl within

Figure 21.2.7 Detection of reactive oxygen species (ROS) with 3-(p-hydroxyphenyl) uores-cein (HPF, H36004) and 3-(p-aminophenyl) uorescein (APF, A36003).

COO

OO O

XH

COO

OO O

OX

ROS

Nonuorescent Fluorescent

X=O 3-(p-hydroxyphenyl) uorescein (HPF)3-(p-aminophenyl) uorescein (APF)X=NH

Figure 21.2.4 N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE, E3101).

N

CH3O

CH2 C OCH2CH3O

Br







the phagovacuole.51,52 OCl also reacts with uorescein (F1300, Section 1.5) to yield uorescent products,53 permitting analysis of OCl levels in water.

Alternatively, 3-(p-aminophenyl) uorescein (APF) and 3-(p-hydroxyphenyl) uorescein (HPF) (A36003, H36004; Section 18.2) can be used for the selective detection of OCl. Both of these uorescein derivatives are essentially nonuorescent until they react with the hydroxyl radical (HO) or peroxynitrite anion (ONOO) (Figure 21.2.7). APF will also react with the hy-pochlorite anion (OCl), making it possible to use APF and HPF together to selectively detect hypochlorite anion. In the presence of these specic ROS, both APF and HPF yield a bright green-uorescent product (excitation/emission maxima ~490/515 nm) and are compatible with all uorescence instrumentation capable of visualizing uorescein. 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.54

Premo Halide Sensore uorescent proteinbased 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 physiologi-cal signicance, it follows that defects in their activity can have severe implications, including such conditions as cystic brosis and neuronal degeneration. us, chloride channels represent important targets for drug discovery.55

e Premo Halide Sensor combines a Yellow Fluorescent Protein (YFP) variant sensitive to halide ions with the ecient and noncytopathic BacMam delivery and expression technol-ogy (BacMam Gene Delivery and Expression TechnologyNote 11.1), yielding a highly sensi-tive, robust and easy-to-use tool for eciently screening halide ion channels and transporter modulators in their cellular models of choice. e Premo Halide Sensor is based on the Venus variant of Aequorea victoria Green Fluorescent Protein (GFP), which displays enhanced uores-cence, increased folding, and reduced maturation time when compared with YFP.56 Additional mutations (H148Q and I152L) were made within the Venus sequence to increase the sensitivity of the Venus uorescent protein to changes in local halide concentration, in particular iodide ions.57 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 ows down the concentration gradient into the cells, where it quenches the uorescence of the expressed Premo Halide Sensor protein (Figure 21.2.8). e decrease in Premo Halide Sensor uorescence is directly proportional to the ion ux, and therefore the chloride channel or trans-porter activity. e Premo Halide Sensor shows a similar excitation and emission prole to YFP (Figure 21.2.9) and can be detected using standard GFP/FITC or YFP lter 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.58

Figure 21.2.9 Quenching of Premo Halide Sensor uo-rescence by increasing concentrations of iodide and chlo-ride. U2OS cells were transduced with Premo Halide Sensor. After 24 hours, cells were trypsinized and lysed by resuspension in sterile distilled water. Fluorescence quench-ing of the lysate was examined using increasing concentra-tions of NaCl (A) and NaI (B). Iodide induces substantially greater quenching of Premo Halide Sensor uorescence than chloride.

Figure 21.2.8 Principle of Premo Halide Sensor Sensor (P10229): Iodide redistribution upon chloride channel activation. Basal uorescence from Premo Halide Sensor is high when chlo-ride channels are low. Upon activation (opening) of chloride channels, the iodide ions enter the cell, down its concentration gradient, and quench the uorescence from Premo Halide Sensor.

Ion channel Ion channelClosed

Open

ActivatedResting

IodidePremo

Halide Sensor

Extracellular Intracellular Extracellular Intracellular

Wavelength (nm)500 600550 650

Fluo

resc

ence

em

issi

on

(arb

itrar

y un

its)

50

100

150

200

250

0

NaCl 0 mM

NaCl 100 mM

NaCl 500 mM

Nal 0 mM

Nal 20 mM

Nal 60 mM

Nal 100 mM

Nal 300 mM

Nal 500 mM

Wavelength (nm)500 600550 650

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(arb

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its)

50

100

150

200

0

A

B








e Premo Halide Sensor (P10229) is pre-packaged and ready for im-mediate use. It contains all components required for cellular delivery and expressionincluding the baculovirus carrying the genetically encoded bi-osensor, BacMam enhancer and stimulus buer containing iodidein ten 96- or 384-well plates. e Premo Halide Sensor has been demonstrated to transduce multiple cell lines including BHK, U2OS, HeLa, CHO, and pri-mary human bronchial epithelial cells (HBEC), providing the exibility to assay chloride-permeable channels in a wide range of cellular models. More information is available at www.invitrogen.com/handbook/premohalide.

Cyanide Detectione homologous aromatic dialdehydes, o-phthaldialdehyde 59 (OPA,

P2331MP) and naphthalene-2,3-dicarboxaldehyde 60 (NDA, N1138), are essentially nonuorescent until reacted with a primary amine in the pres-ence of excess cyanide or a thiol, such as 2-mercaptoethanol, 3-mercapto-propionic acid or the less obnoxious sulte,61 to yield a uorescent isoindole (Figure 21.2.10, Figure 21.2.11). Modied protocols that use an excess of an amine and limiting amounts of other nucleophiles permit the determina-tion of cyanide in blood, urine and other samples.6265

We also oer 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 uorescent isoindoles 6674 (Figure 21.2.12). e 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 iol and Sulde Quantitation Kit (T6060, 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, suldes, sultes and other reducing agents.

Nitrite, Nitrate and Nitric Oxide DetectionWith the discovery of the role of nitric oxide in signal transduc-

tion (Section 18.3), assays for nitrite (NO2) have assumed new impor-tance. 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.

Measure-iT High-Sensitivity Nitrite Assay Kite Measure-iT High-Sensitivity Nitrite Assay Kit (M36051) pro-

vides an easy and accurate method for quantitating nitrite. is kit has an optimal range of 20500 picomoles nitrite (Figure 21.2.13), making it up to 50 times more sensitive than colorimetric methods utilizing the Griess reagent. Nitrates may be analyzed aer quantitative conversion to nitrites through enzymatic reduction.75

Each Measure-iT High-Sensitivity Nitrite Assay Kit contains:

Measure-iT nitrite quantitation reagent (100X concentrate in 0.62 M HCl) Measure-iT nitrite quantitation developer (2.8 M NaOH) Measure-iT nitrite quantitation standard (11 mM sodium nitrite) Detailed protocols

Figure 21.2.12 Fluorogenic amine-derivatization reaction of CBQCA (A6222, A2333).

CBQCA

NNR

CN

COOH

N

C

CHO

COOH

O

+ CN_

R NH2+

Figure 21.2.13 Linearity and sensitivity of the Measure-iT high-sensitivity nitrite assay. Triplicate 10 L 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 uores-cence was not subtracted. The variation (CV) of replicate samples was





Figure 21.2.14 Principle of nitrite quantitation using the Griess Reagent Kit (G7921). Formation of the azo dye is detected via its absorbance at 548 nm.

HO3 NH2 NHH22NH2

NO2

HO3 N2 NNHO3 NHH22NH2

Simply dilute the reagent 1:100, load 100 L into the wells of a microplate, add 110 L sample volumes and mix. Aer a 10-minute incubation at room temperature, add 5 L of de-veloper and read the uorescence. e assay signal is stable for at least 3 hours, and common contaminants are well tolerated in the assay. e Measure-iT High-Sensitivity Nitrite Assay Kit provides sucient material for 2000 assays, based on a 100 L assay volume in a 96-well mi-croplate format; this nitrite assay can also be adapted for use in cuvettes or 384-well microplates.

Griess Reagent KitUnder physiological conditions, NO is readily oxidized to NO2 and NO3 or it is trapped

by thiols as an S-nitroso adduct. e Griess reagent provides a simple and well-characterized colorimetric assay for nitritesand nitrates that have been reduced to nitriteswith a detec-tion limit of about 100 nM.7577 e Griess assay is suitable for measuring the activity of nitrate reductase in a microplate.78 Nitrite reacts with the Griess reagent to form a purple azo derivative that can be monitored by absorbance at 548 nm (Figure 21.2.14).

e Griess Reagent Kit (G7921) contains all of the reagents required for NO2 quantitation, including:

N-(1-Naphthyl)ethylenediamine dihydrochloride Sulfanilic acid in 5% H3PO4 A concentrated nitrite quantitation standard for generating calibration curves Detailed protocols for spectrophotometer- and microplate readerbased assays

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 re-agent. 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.79 NO that has been trapped as an S-nitroso derivative can also be analyzed with the Griess Reagent Kit aer rst releasing the NO from its complex using mercuric chloride or copper (II) acetate.80,81

DAF-FM ReagentDAF-FM 82 (4-amino-5-methylamino-2 ,7-diuorouorescein, D23841; Figure 21.2.15) and

its diacetate derivative (DAF-FM diacetate, D23842, D23844; Section 18.3) have signicant utility for measuring nitric oxide and nitrite production in live cells and solutions. e uo-rescence quantum yield of DAF-FM is reported to be 0.005 but increases about 160-fold to 0.81 aer reacting with nitrite 82 (Figure 21.2.16). DAF-FM has some important advantages over the similar nitric oxide sensor, DAF-2, and other aromatic diamines:

Spectra of the NO (NO2) adduct of DAF-FM are independent of pH above pH 5.5.82

NO2 adduct of DAF-FM is signicantly more photostable than that of DAF-2.82

DAF-FM is a more sensitive reagent for NO2 than is DAF-2; the NO and NO2 detection limit for DAF-FM is ~3 nM 82 versus ~5 nM for DAF-2.83

e higher absorptivity and greater water solubility of the NO2 adduct of DAF-FM should make this assay much more sensitive than detection with 2,3-diaminonaphthalene or other aromatic diamines.

Figure 21.2.16 Fluorescence emission spectra of DAF-FM (D23841, D23842, D23844) in solutions containing 01.2 M nitric oxide (NO).

Fluo

resc

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em

issi

on

Wavelength (nm)500 525 550 600

1.2 M NO

1.1

475 575

0.89

0.71

0.54

0.36

0.180

Figure 21.2.15 DAF-FM (4-amino-5-methylamino-2,7-diuoro-uorescein, D23841).








Figure 21.2.18 Principle of the PiPer Phosphate Assay Kit (P22061). In the presence of inorganic phosphate, maltose phos-phorylase converts maltose to glucose 1-phosphate and glucose. Then, glucose oxidase converts the glucose to gluconolac-tone and H2O2. Finally, with horseradish peroxidase (HRP) as a catalyst, the H2O2 reacts with the Amplex Red reagent to gen-erate the highly uorescent resorun. The resulting increase in uorescence or absorption is proportional to the amount of Pi in the sample.

N

O OHHO

C CH3O

N

O OHO

O

HO

CH2OH

O

OH

OHO

HO

CH2OH

OHOH

OHO

HO

CH2OH

OHO P

O

O

O

OHO

CH2OH

OHOH

OOH

CH2OH

HO

OOH

+

H2O2O2

Phosphate (Pi) +Maltose phosphorylase

Resorun(uorescent)

Glucose oxidase

HRP

OH

Amplex Red(nonuorescent)

Figure 21.2.19 Detection of inorganic phosphate using the PiPer Phosphate Assay Kit (P22061). Each reaction contained 50 M 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 buer. Reactions were incu-bated at 37C. After 60 minutes, uorescence was measured in a uorescence microplate reader using excitation at 530 12.5 nm and uorescence 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.

2500

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01.60.40 0.8

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1000

40

Because the reaction of DAF-FM with NO requires a preliminary nonspecic oxidation step, it is important to also perform control experiments with nitric oxide synthase inhibitors to conrm the source of the uorescent species.84

2,3-DiaminonaphthaleneWe also oer 2,3-diaminonaphthalene (D7918, Figure 21.2.17), which reacts with NO2 to

form the uorescent product 1H-naphthotriazole. A rapid, quantitative uorometric assay that employs 2,3-diaminonaphthalene can reportedly detect from 10 nM to 10 M NO2, and is com-patible with a 96-well microplate format.85 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.86 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.86

NBD MethylhydrazineNBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan, M20490) has been

used to measure NO2 in water.87 Reaction of NBD methylhydrazine with NO2 in the presence of mineral acids leads to formation of uorescent products with excitation/emission maxima of ~468/537 nm. is reaction serves as the principle behind a selective uorogenic method for the determination of NO2. e assay is suitable for measurements by absorption or uorescence spectroscopy or by uorescence-detected HPLC.87

Other Nitrate Detection ReagentsRhodamine 110 (R6479) has proven useful in a uorescence quenching method for deter-

mining trace nitrite.88 is sensitive assay takes advantage of the reaction of the green-uo-rescent rhodamine 110 with nitrite at acidic pH to form a nitroso product that exhibits much weaker uorescence. With a linear range of 1 108 to 3 107 moles/L and a detection limit of 7 1010 moles/L, this assay has been used to measure nitrite in tap water and lake water without any prior extraction procedures.

Ecient quenching of SPQ or MQAE uorescence (M440, E3101; see above) by nitrite (but not nitrate) has been used for direct measurement of NO2 transport across erythrocyte mem-branes 89 and for functional assays of bacterial nitrite extrusion transporters.90

Figure 21.2.17 2,3-diaminonaphthalene (D7918).

NH2

NH2







Phosphate and Pyrophosphate DetectionPiPer Phosphate Assay Kit

e PiPer Phosphate Assay Kit (P22061) provides an ultrasensitive assay that detects free phosphate in solution through formation of the uorescent product resorun. Because resorun also has strong absorption, the assay can be performed either uorometrically or spectrophoto-metrically. is 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 ki-nase. Furthermore, the assay can be modied to detect virtually any naturally occurring organic phosphate molecule by including an enzyme that can specically digest the organic phosphate to liberate inorganic phosphate.

In the PiPer phosphate assay (Figure 21.2.18), maltose phosphorylase converts maltose (in the presence of Pi) to glucose 1-phosphate and glucose. en glucose oxidase converts the glu-cose 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 reso-run, which has absorption/emission maxima of ~571/585 nm.91,92 e resulting increase in uorescence or absorption is proportional to the amount of Pi in the sample. is kit can be used to detect as little as 0.2 M Pi by uorescence (Figure 21.2.19) or 0.4 M Pi by absorption.

e PiPer Phosphate Assay Kit contains:

Amplex Red reagent Dimethylsulfoxide (DMSO) Concentrated reaction buer Recombinant maltose phosphorylase from Escherichia coli Maltose Glucose oxidase from Aspergillus niger Horseradish peroxidase Phosphate standard Hydrogen peroxide Detailed protocols for detecting phosphatase activity

Each kit provides sucient reagents for approximately 1000 assays using a reaction volume of 100 L per assay and either a uorescence or absorbance microplate reader.

PiPer Pyrophosphate Assay Kite PiPer Pyrophosphate Assay Kit (P22062) provides a sensitive uorometric or colori-

metric 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 pyrophos-phate assay, inorganic pyrophosphatase hydrolyzes PPi to two molecules of inorganic phosphate (Pi). e Pi then enters into the same cascade of reactions as it does in the PiPer Phosphate Assay Kit (Figure 21.2.18). In this case, the resulting increase in uorescence or absorption is proportional to the amount of PPi in the sample. is kit can be used to detect as little as 0.1 M PPi by uorescence or 0.2 M PPi by absorption (Figure 21.2.20).

e PiPer Pyrophosphate Assay Kit contains:

Figure 21.2.20 Detection of pyrophosphate using the PiPer Pyrophosphate Assay Kit (P22062). Each reaction contained 50 M Amplex Red reagent, 0.01 U/mL inorganic pyrophosphatase, 2 U/mL maltose phosphorylase, 0.2 mM maltose, 1 U/mL glucose oxidase and 0.2 U/mL HRP in 1X reaction buer. Reactions were incubated at 37C. After 60 minutes, A) uorescence was measured in a uores-cence-based microplate reader using excitation at 530 12.5 nm and uorescence detection at 590 17.5 nm or B) absorbance was measured in an absorption-based micro-plate reader at 576 5 nm. Data points represent the aver-age of duplicate reactions. In panel A, a background value of 78 (arbitrary units) was subtracted from each reading; in panel B, a background absorbance of 0.011 was subtracted from each reading.

0 80

Ab

sorb

ance

0.3

0.0

40 6020

0.1

0.2

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100 120

0.04

0.08

0.06

0.02

0 4 8 120.00

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15,000

0

20,000

25,000

10,000

5,000

10,000

5,000

0 1 32 4

0

25,000

20,000

15,000

A

B

Amplex Red reagent Dimethylsulfoxide (DMSO) Concentrated reaction buer Recombinant maltose phosphorylase

from Escherichia coli Maltose Glucose oxidase from Aspergillus niger

Horseradish peroxidase Inorganic pyrophosphatase from

bakers yeast Pyrophosphate standard Detailed protocols for detecting pyro-

phosphatase activity

Each kit provides sucient reagents for approximately 1000 assays using a reaction volume of 100 L per assay and either a uorescence or absorbance microplate reader.


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Indicators for Na+, K+, Cl– and Miscellaneous Ions