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].

Indo-1 AM is a fluorescent Ca2+ indicator (λex=340 nm, λem=405/485 nm).

Indo-1 AM is a fluorescent Ca2+ indicator with λex=340 nm and λem=405/485 nm[1]. In rabbit hearts that are exposed to Indo-1 AM, 72% of the dye is in the cytosol, and the remainder is associated with the mitochondria, microsomes and the nucleus[2].

[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. [2]. Blatter LA, et al. Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2. Biophys J. 1990 Dec;58(6):1491-9.