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/) FDSS is registered trademark of Hamamatsu Photonics K.K. (China, France, Germany, Italy, Japan, U.K., U.S.A.) Product and software package names noted in this documentation are trademarks or registered trademarks of their respective manufacturers. • Subject to local technical requirements and regulations, availability of products included in this promotional material may vary. Please consult your local sales representative. • Information furnished by HAMAMATSU is believed to be reliable. However, no responsibility is assumed for possible inaccuracies or omissions. 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