Ix71 live cell imaging fluorescence microscope

Intra/interlobular ducts were isolated from the pancreas of WT and AQP KO mice using the microdissection technique as described previously (Argent et al., 1986 (link)). Changes in intracellular pH (pH_i) were detected using the pH-sensitive fluorescence dye, BCECF. Pancreatic ducts were incubated with BCECF-AM (2 μM) for 30–60 min, at room temperature. After the incubation, ducts were attached to a cover glass, which formed the base of a perfusion chamber, mounted on the stage of an IX71 live cell imaging fluorescence microscope (Olympus, Budapest, Hungary) and excited at 440 and 490 nm. Emissions were monitored at 530 nm. Five to seven region of interests (ROIs) were examined in each experiment, and one measurement per second was obtained. The 490/440 fluorescence ratio was calibrated to pH_i using the high K⁺-nigericin technique, as previously described (Thomas et al., 1979 (link); Hegyi et al., 2004 (link)).

The [Ca²⁺]_i determination protocols have been described previously (Yan et al., 2013 (link)). Briefly, in order to determine [Ca²⁺]_i, an Olympus IX71 live cell imaging fluorescence microscope (Tokyo, Japan) was used to record the fluorescence intensity of fura-2 AM in saline solution (NaCl 137 mM; KCl 5.4 mM; KH₂PO₄ 0.44 mM; NaHCO₃ 4 mM; CaCl₂ 1.3 mM; MgSO₄ 0.8 mM; MgCl₂ 5 mM, and glucose 10 mM, pH 7.4) for 30 min at 37°C for glioma cells grown on coverslips coated with polylysine. After two washes with this saline solution, coverslips were either perfused in the saline solution (control), or TMZ was carefully added to the saline at time zero. Readings were taken at 340 and 380 nm excitation and 510 nm emission at 20-s intervals. The incubation continued for 22 min. Between 13 and 20 cells were selected for each coverslip, and three to five coverslips were assessed in each experimental group. The represents of this experiment performed means from 38 to 81 cells on three to five individual coverslips of each treatment.

For [Ca²⁺]_i recordings an Olympus IX71 live cell imaging fluorescence microscope (Tokyo, Japan) was used to monitor fluorescence intensity of Fura-2 loaded cultured neurones. For Fura-2/AM loading the growth medium was replaced with saline solution (137 mM NaCl, 5 mM KCl, 0.44 mM KH₂PO₄, 4 mM NaHCO₃, 1.3 mM CaCl₂, 0.8 mM MgSO₄, and 0.5 mM MgCl₂ with 10 mM glucose) containing 5 μM Fura-2/AM for 30 min at 37°C. After two times wash with similar saline, the coverslip was perfused with the saline solution, and recordings were made at 340 and 380 nm excitation and 510 nm emission wavelengths at 15 s intervals. The data are presented as 340/380 ratio R normalized to 340/380 ratio reading at the rest (R₀). The SOCE was assessed by measuring the amplitude of [Ca²⁺]_i transient induced by re-addition of extracellular Ca²⁺ to cells pre-incubated with Ca²⁺-free buffer in combination with the SERCA inhibitor thapsigargin. Twenty cells were selected in each coverslip, and three coverslips were averaged in each experimental group.