CHAPTER 21
Indicators for Na+, K+, Cl and Miscellaneous Ions
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TW
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E
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
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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.
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Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
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The Molecular Probes Handbook: A Guide to Fluorescent Probes and
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this manual are covered by one or more Limited Use Label
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Product List on page 975. Products are For Research Use Only. Not
intended for any animal or human therapeutic or diagnostic use.
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
resc
ence
exc
itatio
n
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
resc
ence
exc
itatio
n
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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Labeling Technologies
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Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
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The Molecular Probes Handbook: A Guide to Fluorescent Probes and
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this manual are covered by one or more Limited Use Label
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Product List on page 975. Products are For Research Use Only. Not
intended for any animal or human therapeutic or diagnostic use.
Section 21.1 Fluorescent Na+ and K+ Indicators
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
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.thermofisher.com/probes
Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
907www.invitrogen.com/probes
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
Section 21.1 Fluorescent Na+ and K+ Indicators
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
resc
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
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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.
Section 21.1 Fluorescent Na+ and K+ Indicators
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
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling Technologies
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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.
Section 21.1 Fluorescent Na+ and K+ Indicators
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
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The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
Section 21.1 Fluorescent Na+ and K+ Indicators
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
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Section 21.1 Fluorescent Na+ and K+ Indicators
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).
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REFERENCES
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.
thermofisher.com/probes
Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
912www.invitrogen.com/probes
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
Section 21.1 Fluorescent Na+ and K+ Indicators
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
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Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
913www.invitrogen.com/probes
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
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)
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Labeling Technologies
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thermofisher.com/probes
Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
914www.invitrogen.com/probes
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
Section 21.2 Detecting Chloride, Phosphate, Nitrite and Other
Anions
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 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.
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human therapeutic or diagnostic use.thermofisher.com/probes
Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
915www.invitrogen.com/probes
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
Section 21.2 Detecting Chloride, Phosphate, Nitrite and Other
Anions
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
Fluo
resc
ence
em
issi
on
(arb
itrar
y un
its)
50
100
150
200
0
A
B
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thermofisher.com/probes
Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
916www.invitrogen.com/probes
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
Section 21.2 Detecting Chloride, Phosphate, Nitrite and Other
Anions
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
Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
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Labeling TechnologiesIMPORTANT 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.
Section 21.2 Detecting Chloride, Phosphate, Nitrite and Other
Anions
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
ence
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).
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.
thermofisher.com/probes
Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
918www.invitrogen.com/probes
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
Section 21.2 Detecting Chloride, Phosphate, Nitrite and Other
Anions
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
2000
500
0
Phosphate (M)
Fluo
resc
ence
1008040 60200
01.60.40 0.8
120
1.2
80
1500
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
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.thermofisher.com/probes
Chapter 21 Indicators for Na+, K+, Cl and Miscellaneous Ions
919www.invitrogen.com/probes
The Molecular Probes Handbook: A Guide to Fluorescent Probes and
Labeling TechnologiesIMPORTANT 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.
Section 21.2 Detecting Chloride, Phosphate, Nitrite and Other
Anions
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
Pyrophosphate (M)
100 120
0.04
0.08
0.06
0.02
0 4 8 120.00
Fluo
resc
ence
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.
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). Pl