Paleal|a|8eas|l|re ANfP Dyes NP û1199

hsr/ss4. 1b/s//es/,2001
Praáacl |aIarmal|aa
PeteatialSeasitive 4¥IP 0,es
latredactiea
The ANEP (aminonaphthylethenylpyridinium) dyes
developed by Leslie Loew and colleagues
1,2
are the most consis-
tently sensitive probes for detection of submillisecond membrane
potential changes. Di-4-ANEPPS (D-1199) has a fairly uniform
10% per 100 mV change in fluorescence intensity in a variety of
tissue, cell and model membrane systems.
3
ANEP dyes undergo
changes in their electronic structure, and consequently their fluo-
rescence spectra, in response to changes in the surrounding elec-
tric field.
2
This optical response is sufficiently fast to detect
transient potential changes in excitable cells, including single
neurons, cardiac cells and intact tissue preparations. Further-
more, these dyes display a potential-dependent shift in their exci-
tation spectra, thus permitting the quantitation of membrane po-
tential using excitation ratio measurements.
4
Structural
variations among the ANEP dyes confer suitability for special-
ized applications (Figure 1, Table 1).
Sterage aad haadliag
These products are provided as lyophilized solids and should
be stored desiccated and protected from light at room tempera-
ture until use. Generally suitable stock solutions can be made in
ethanol, DMSO or DMF at 1 mg/mL (approximately 2 mM);
store stock solutions protected from light at 4°C.
Sµectral 0baracteristics
The spectral characteristics of ANEP dyes are all gene-
rally similar. Their maximum molar extinction coefficients for
absorption in methanol are about 36,000 cm
-1
M
-1
at about
498 nm (for the zwitterionic ANEPPS dyes) or 517 nm (for the
cationic ANEPEQ and ANEPPQ dyes). Absorption and fluores-
cence spectra of the ANEP dyes are highly dependent on their
environment.
1,9
The dyes are essentially nonfluorescent in water
and become quite strongly fluorescent upon binding to mem-
branes. The absorption and fluorescence emission maxima of di-
8-ANEPPS bound to model phospholipid
membranes are about 465 nm and 635 nm, respectively (full
Figure 1. Structures of ANEP dyes.
Table 1. Functional characteristics of ANEP dyes.
Cat # Name Characteristics Ref

*
D-1199 di-4-ANEPPS Most consistent potentiometric
response in different cell and
tissue types
3
D-3167 di-8-ANEPPS Less susceptible to internali-
zation than di-4-ANEPPS,
permitting extended
observation
5
D-6923 di-2-ANEPEQ

† Highly water-soluble ANEP
dye for microinjection
6
D-6925 di-8-ANEPPQ Most sensitive potentiometric
response in retrogradely
labeled neurons
7,8
D-6927 di-12-ANEPPQ Similar to di-8-ANEPPQ;
useful for retrograde labeling
of neurons
7,8
* Numbers refer to the main reference list below. † Also known as
JPW 1114.
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Paleal|a|8eas|l|re ANfP Dyes 2
spectra can be found at our Web site, www.probes.com). Fluo-
rescence is insensitive to pH and photostability is generally high.
4µµlicatiea
0ellalar labeliag
1. Bulk Loading. Cells attached to coverslips are typically
labeled by direct addition of a small amount of dye stock solution
in DMSO to the cell incubation medium to produce a loading
concentration ranging from 0.2–2 µM. Higher dye concentra-
tions (10–50 µM) are used for loading perfused tissue
samples.
10,11
Pluronic
®
F-127 (0.05%) is often included
in the loading solution to aid the solubilization of the dye (this
is particularly necessary with the more lipophilic ANEP dyes
such as di-8-ANEPPS). Molecular Probes offers Pluronic
F-127 in three forms: 1 mL of a 20% (w/v) solution in DMSO
(P-3000), 30 mL of a sterile 10% (w/v) solution in water
(P-6866) and 2 g solid (P-6867). Low incubation temperatures
(4–20ºC) inhibit internalization of the dye. After incubation for
10–20 minutes in the presence of the dye, cells are washed up to
three times in dye-free medium.
2. Retrograde Labeling. Specific groups of neurons can be
labeled by localized pressure injection of a concentrated dye so-
lution (10–20 mg/mL in DMSO or ethanol) into neuronal
tissue. Retrograde labeling of cell bodies with di-8-ANEPPQ
helereaces
1. Biochemistry 24, 5749 (1985); 2. Pure Appl Chem 68, 1405 (1996); 3. J Membr Biol 130, 1 (1992); 4. Biochemistry 28, 4536 (1989);
5. Neuron 9, 393 (1992); 6. J Neurosci 15, 1392 (1995); 7. J Neurosci Methods 70, 111 (1996); 8. J Neurosci Methods 70, 121 (1996);
9. Biochim Biophys Acta 1323, 223 (1997); 10. J Neurosci 16, 3456 (1996); 11. Am J Physiol 270, H2216 (1996).
Predact list le//set c//css ms, /s e/ts/es4 //em ee/ Ws/ s/ts e/ //em ee/ lestems/ Ss/r/cs 0scs/tmset.
Cat # Product Name Unit Size
D-6923 di-2-ANEPEQ (JPW 1114) ....................................................................................................................................................... 5 mg
D-6925 di-8-ANEPPQ ............................................................................................................................................................................ 5 mg
D-6927 di-12-ANEPPQ .......................................................................................................................................................................... 5 mg
D-1199 di-4-ANEPPS ............................................................................................................................................................................ 5 mg
D-3167 di-8-ANEPPS ............................................................................................................................................................................ 5 mg
P-6867 Pluronic
®
F-127 *low UV absorbance* ...................................................................................................................................... 2 g
P-3000 Pluronic
®
F-127 *20% solution in DMSO* ................................................................................................................................. 1 mL
P-6866 Pluronic
®
F-127 *sterile 10% solution in water* ........................................................................................................................ 30 mL
V-1644 valinomycin................................................................................................................................................................................ 25 mg
(D-6925) in chicken spinal cord was found to occur within
24 hours.
7
3. Microinjection. Microelectrode tips filled with a 3 mg/mL
(5.5 mM) aqueous solution of di-2-ANEPEQ (D-6923) can be
used for pressure microinjection into neurons.
6
The dye solution
should be filtered immediately before filling to remove micro-
scopic particles that may cause blockage of the electrode tip.
llaeresceace Neasaremeats
Fluorescence ratio imaging measurements are typically per-
formed by recording fluorescence intensities excited at about 450
and 510 nm, detecting emission at >570 nm.
3,4
The ratio of
these intensities (F
450
/F
510
) decreases upon membrane hyper-
polarization. Potential-dependent fluorescence emission ratio
measurements (ratio of emission intensities at 560 nm and
620 nm, excitation at 475 nm) have also been reported using di-
8-ANEPPS.
11
Calibration of the potentiometric response can be
accomplished using the ionophore valinomycin
(V-1644) and varying membrane potential with the addition
of different potassium concentrations to the extracellular
medium.
3,4
Paleal|a|8eas|l|re ANfP Dyes 3
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