Rhod 2

The hiPSC-CMs were seeded onto fibronectin-coated thin glass bottom (0.085–0.115 mm) 35 mm dish (In Vitro Scientific) and treated with ETP for 48 h. Then, the cells were washed and incubated with 2 µM Rhod-2, AM Ca²⁺ dye (Invitrogen) for 30 min at 37 °C, and washed. Rhod-2-labeled cells were then observed and fluorescence imaging was performed at room temperature, with an inverted confocal laser-scanning microscope (FV1000, Olympus) operating in the line-scan mode, equipped with a 60× oil immersion objective. Upon recording a line scans, background subtraction was applied for all data sets, by defining the average (auto-) fluorescence intensity of an extracellular area as the background. The data were then subjected to a fitting procedure which adjusted the parameters of a Weibull function implemented in Sigma Plot (Version 8.04, SPSS). A self-made macro in Excel (Microsoft) calculated the Ca²⁺ transient amplitude (F/F₀, where F₀ is the averaged background-corrected resting fluorescence intensity), time-to-peak (TTP), the maximum steepness ([ΔF/ΔT] max), full-width at half-maximum (FWHM) and T90%. Data were represented as ± SEM of 25 measurement (n = 25) for each group.

Brain tissues were immersed in ice-cold ACSF solution, consisting of 124 mM NaCl, 1.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 1.25 mM NaH₂PO₄, 22 mM NaHCO₃, and 10 mM glucose. Brain slices (350 μm) were incubated in the ACSF (pH 7.3) equilibrated with 95% O₂ to 5% CO₂ for 30 min at room temperature and loaded with a fluorescent Ca²⁺ indicator, 10 mM rhod2-AM (Dojindo, Kumamoto, Japan), for 90 min. rhod2-AM-loaded slices were placed in a small flow-through chamber on a microscope stage and continuously perfused (2 mL/min) with the equilibrated ACSF solution at 32°C . Fluorescence images of rhod-2-loaded slices (>580 nm), at the excitation wavelength of 520–550 nm were obtained through a low-magnification objective lens (UM Plan Fl 10×, Olympus, Tokyo, Japan). Data were recorded using the imaging system as described previously with modifications.³³ (link) Images were sequentially captured every 20 s. For quantitative analysis, the fluorescence intensity at each time point was divided by that of the initial image stored as a reference.