Hcimage system

The membrane potential was measured using the fluorescence voltage‐sensitive dye DiBAC₄(3), as previously reported.²⁷ (link) The cells were continuously incubated with 100 nmol/L DiBAC₄(3) throughout the experiments. In membrane potential imaging, cells loaded with DiBAC₄(3) were illuminated at a wavelength of 490 nm. The intracellular Ca²⁺ concentrations were measured using the fluorescent Ca²⁺ indicator dye Fura 2‐AM (DOJINDO). Cells were incubated with 10 μmol/L Fura 2‐AM for 30 min at room temperature. Cells loaded with Fura 2 were alternatively illuminated at wavelengths of 340 and 380 nm. Fluorescence images were recorded on an ORCA‐Flash2.8 digital camera (Hamamatsu Photonics). Data collection and analyses were performed using an HCImage system (Hamamatsu Photonics). Images were measured every 5 s, and the values of fluorescence intensity (F) were determined by measuring the average for 1 min (12 images). The fluorescence intensity of Fura 2 was expressed as measured 340/380 nm fluorescence ratios after background subtraction.

Cells were seeded onto 35 mm glass bottom dishes and cultured at 37°C in 5% CO₂ humidified incubator. Intracellular Ca²⁺ concentration was measured using the fluorescent Ca²⁺ indicator dye, Fura 2‐AM. Cells were incubated with 10 μmol/L Fura 2‐AM in normal HEPES solution for 30 min at room temperature. Cells loaded with Fura 2‐AM were alternatively illuminated at wavelengths of 340 and 380 nm, and fluorescence images were recorded on the ORCA‐Flash2.8 digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Data collection and analyses were performed using an HCImage system (Hamamatsu Photonics). Images were measured every 5 sec. The fluorescent intensity of Fura 2 was expressed as measured 340/380 nm fluorescence ratios [ratio (340/380)] after background subtraction. After treatment with a sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) inhibitor, thapsigargin (1 μmol/L) under 0 mmol/L Ca²⁺ in a bathing medium, application of 2.2 mmol/L Ca²⁺ caused store‐operated Ca²⁺ entry (SOCE) (see Fig. S3B). Ten minutes after 1st application of 2.2 mmol/L Ca²⁺, second one was performed in the presence of 1 mmol/L TRAM‐34. The value of area under the curve (AUC) of the ratio (340/380) was obtained by the integration on the computer. The AUC value in the second application was normalized by that of the first one (relative AUC of [Ca²⁺]_i).