Hcimage

Changes in intracellular calcium were measured with the fluorescent calcium indicator, Fura-2, as previously described (Masuoka et al., 2015 (link), 2017 (link)). For the microscopic fluorometric measurement, cultured trigeminal ganglion neurons were washed twice with Krebs–Henseleit solution and incubated for 1 h in a solution containing 3 μM of Fura-2-acetoxymethyl ester (Fura-2 AM; Dojindo Laboratories, Kumamoto, Japan) and 0.005% Cremophor EL (Sigma–Aldrich). After incubation, the cells were washed in Krebs–Henseleit solution for 30 min, and culture dishes were placed on the stage of an inverted microscope (ECLIPSE TE 300, Nikon, Tokyo, Japan) equipped with a 20× S-fluor objective. Fluorescence images were recorded and analyzed using a video image analysis system (HCimage, Hamamatsu Photonics, Hamamatsu, Japan). The experimental agents were dissolved in Krebs–Henseleit solution and delivered by continuous perfusion in the recording chamber with a peristaltic pump (2 ml/min). The perfused solutions were maintained at 34°C with a temperature controller (TC-344C and SH-27B, Warner Instruments). Image pairs of Fura-2 fluorescence were captured every 10 s at an emission wavelength of 510 nm by exciting Fura-2 at 340 and 380 nm. The 340–380 nm fluorescence ratio (F340/F380) was used as a parameter of intracellular calcium concentration.