A cell-permeant precursor of Indo-1
Indo-1 AM is a cell-permeant acetoxymethyl ester of the ratiometric fluorescent calcium indicator indo-1 . As indo-1 AM enters cells, it is hydrolyzed by intracellular esterases to produce Indo-1. Indo-1 is ideal for analyses using flow cytometry, as it uses a single excitation source, typically 349-364 nm light from an argon-ion laser. The emission maximum shifts from 475-485 nm without calcium to 400-410 nm when indo-1 binds calcium.1,2 Indo-1 is prone to photobleaching, which limits its usefulness in methods involving microscopy.1
1.Paredes, R.M., Etzler, J.C., Watts, L.T., et al.Chemical calcium indicatorsMethods46(3)143-151(2008) 2.Grynkiewicz, G., Poenie, M., and Tsien, R.Y.A new generation of Ca2+ indicators with greatly improved fluorescence propertiesJ. Biol. Chem.260(6)3440-3450(1985)
Cell experiment: | The cells are loaded with indo-I and fura-2 by exposure to the membrane-permeant forms, Indo-1 AM (indo-l-acetoxymethyl ester) and fura-2-acetoxymethyl ester, respectively. A mixture of 10 μL of 1 mM Indo-1 AM or fura-2/AM in dimethylsulfoxide (DMSO), 2.5 μL of 25% wt/wt Pluronic F-127 in DMSO, and 75 μL fetal calf serum (FCS) or newborn calf serum (NCS) is added to 2 mL Tyrode solution containing the cells and 40 μL FCS or NCS. Loading is achieved by gently shaking the cells for 15 to 30 min[2]. |
参考文献: [1]. Andrienko T, et al. Real-Time Fluorescence Measurements of ROS and [Ca2+] in Ischemic / Reperfused Rat Hearts: Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning. PLoS One. 2016 Dec 1;11(12):e0167300. | |
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