FDSS Application Note No.2 CoroNa Red as a potential probe for

FDSS Application Note No.2
FDSS7000EX
CoroNa Red as a potential probe
for intracellular sodium imaging
Abstract
We have examined the new sodium indicator fluorescent
dye named CoroNa Red using FDSS. Molecular Probe
newest Na+ indicator is CoroNa Red chloride, which is
based on a crown ether that has structural similarity to the
Ca2+ chelator BAPTA. Unlike SBFI and Sodium Green, the
net positive charge of CoroNa Red targets the indicator to
mitochondria and loading of cells does not require use of a
permeant ester derivative of the dye. CoroNa Red is only
weakly fluorescent in the absence of Na + and its
fluorescence increases ~15-fold upon binding Na+. Despite
its relatively high Kd for Na + of ~200 mM (Table. 1),
CoroNa Red indicator exhibits sensitive responses to
cellular Na+ influxes through voltage-gated channels and
ATP-gated cation pores1),2),3). Here we present the first
data for the use of CoroNa Red with the FDSS "Functional
Drug Screening System" (Hamamatsu Photonics K. K.,
Hamamatsu City, Japan).
Table. 1: Comparison data of each sodium indicator.
MW
Molecular Probe Cat
Kd(K+free)
Kd(K+Sat.)
Selectivity for Na+ than K+
Fluorescence quantum yield
Resting cell
Fully depolarized
Excitation wavelength
Emission wavelength
CoroNa Red Sodium Green
773.32
1543
S-6901
C-24430
~ 200 mM
6 mM
21 mM
~ 200 mM
~ 41-fold
0.2
540 nm
590 nm
480 nm
540 nm
SBFI
1127
S-1263
3.8 mM
11 mM
~ 18-fold
0.08
~ 15 mM
~ 25 mM
335 nm
540 nm
Materials and Methods
Results and Discussion
Human smooth muscle cell were tripsinized and plated at
4 × 104 per well. Cells are typically loaded by adding 5 to
10 µM CoroNa Red from a 1 mM stock solution in DMSO,
incubating for 10 to 30 minutes at 37 ˚C and finally
washing with dye-free medium before FDSS analysis. The
light emission was recorded during variable times using
the FDSS. Single-wavelength analysis with CoroNa red
needs the excitation filters 540 nm, the 560 nm dichroic
mirror, and a 590 nm emission filter.
To affirm the Na+ response of CoroNa red, we used the
veratridine/ 3-Veratroylveracevine (Sigma V5754).
Veratridine is one of the neurotoxin recognize the -subunit
(site2) of voltage gated Na + channels 4). As expected,
fluorescent ratios were increased immediately after
stimulation (Figure. 1). These results are basically
consistent with previous data. We present the first data for
the use of sodium indicator CoroNa Red with the FDSS.
The longer-wavelength absorption of the CoroNa Red
indicator results in reduction of the potential for
photodamage to the cell because the energy of the
excitation light is lower than that of the UV light required
for excitation of SBFI. The high Kd for Na+ of ~200 mM,
approximating physiological ionic strength, CoroNa Red
indicator will become the useful dye for imaging the
intracellular sodium influxes.
References
1. Paolo Bernardi, Mitochondrial Transport of Cations: Channels,
Exchangers, and Permeability Transition.
Physiological Reviews, Vol. 79, No. 4, October 1999, pp. 11271155
1.16
2. Jung, DW., et al., Transmembrane gradients of free Na+ in
isolated heart mitochondria estimated using a fluorescent probe.
Am J Physiol 1992 Apr;262(4 Pt 1):C1047-55
1.14
1.12
1.1
Ratio
FDSS Application Note No.2
H a m a m a t s u S c r e e n i n g T e c h n o l o g y
CoroNa Red imaging of Human
smooth muscle cell
3. Dennis W. Jung., et al., The Sodium-Calcium Antiport of Heart
Mitochondria Is Not Electroneutral.
J Biol Chem 1995 Jan 13;270(2):672-8
1.08
1.06
4. McKinney, L.C., et al., Purification, solubility, and pKa of
veratridine. Analyt. Biochem. 153, 33-38 (1986)
1.04
FDSS7000EX
1.02
1
0.98
0
20
40
60
80
Time (s)
Veratridine 1 µM
Veratridine 0.5 µM
Veratridine 0.1 µM
Veratridine 0.05 µM
Consumable
CoroNa™ Red chloride
Molecular Formula: C42H49ClN4O8
Molecular Weight: 773.32
C-24430 CoroNa™ Red chloride 1 mg
C-24431 CoroNa™ Red chloride *special packaging* 20 ×
50 µg
Molecular Probe Cataloge
(http://www.probes.com/)
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