Dissociated hSS (day 62) or hCS (day 123) derived from control and TS lines were cultured on poly-L-ornithine and laminin (Sigma) coated coverslips for 4–5 days. The cultures were incubated with 1 μM Fura-2 acetoxymethyl ester (Fura-2AM; Invitrogen) for 25 min at 37°C in NM medium, washed for 5 min and placed in a perfusion chamber on the stage of an inverted fluorescence microscope (TE2000U; Nikon). Cells were then stimulated with high-KCl Tyrode’s solution (67 mM KCl, 67 mM NaCl2 mM CaCl2, 1 mM MgCl2, 30 mM glucose and 25 mM HEPES, pH 7.4). Imaging was performed at room temperature (25°C) on an epifluorescence microscope equipped with an excitation filter wheel and an automated stage. Openlab software (PerkinElmer) was used to collect and quantify time-lapse excitation ratio images. Fluorescence images were analyzed using the IGOR Pro software (WaveMetrics). Residual calcium following high-KCl depolarization was calculated by dividing the plateau calcium level by the peak calcium elevation ((C – A)/(B – A); Fig. 3b).
