Cfi s fluor 40 oil

Cells were incubated with 5 μM fura-FFP18/AM for 2 h at 37 °C as described previously [45 (link)]. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti2, Amsterdam, The Netherlands) with an image acquisition and analysis system for videomicroscopy (NIS-Elements Imaging Software, Nikon). Cells were continuously superfused with HBS supplemented with 0.1% (w/v) BSA at room temperature and were examined at 40× magnification (Nikon CFI S FLUOR 40× Oil, Amsterdam, The Netherlands). Cells were alternatively excited with light from a xenon lamp passed through a high-speed monochromator Optoscan ELE 450 (Cairn Research; Faversham, UK) at 335 and 364 nm, and fluorescence emission, at 490 and 502 nm, respectively, was detected using a cooled digital sCMOS camera Zyla 4.2 (Andor; Belfast, UK) and recorded using NIS-Elements AR software (Nikon, Tokyo, Japan). Fluorescence ratio (F335/F364) was calculated pixel by pixel, and the data were presented as ΔF₃₃₅/F₃₆₄. TG-evoked Ca²⁺ entry was measured as the integral of the rise in fura-FFP18 fluorescence ratio for 3 min after the addition of extracellular Ca²⁺ taking a sample every second (AUC). To compare the rate of increase in fura-FFP18 fluorescence between different treatments, traces were fitted to the equation mentioned in Section 4.3.

Cells were loaded with fura‐2 by incubation for 30 min with 2 μm fura‐2/AM at 37 °C as described [33 (link), 34 (link)]. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti2, Amsterdam, the Netherlands) with an image acquisition and analysis system for videomicroscopy (nis‐elements Imaging Software v.5.02.00; Nikon, Amsterdam, the Netherlands). Cells were superfused at room temperature with HEPES‐buffered saline (HBS) containing 125 mm NaCl, 5 mm KCl, 1 mm MgCl₂, 5 mm glucose, and 25 mm HEPES, pH 7.4, supplemented with 0.1% (w/v) BSA and examined at 40× magnification (Nikon CFI S FLUOR 40× Oil). Samples were alternatively excited at 340/380 nm as described previously [35 (link)]. Fluorescence ratio (F340/F380) was calculated pixel by pixel, and the data were presented as ΔF340/F380. Thapsigargin (TG)‐evoked rises in [Ca²⁺]_c and SOCE were estimated as the area under the curve measured as the integral of the rise in fura‐2 fluorescence 340/380 nm ratio during 2.5 min after the addition of the agonist or extracellular Ca²⁺, respectively, and taking a sample every 2 s.

Cells were loaded with fura-2 by incubation with 5 μM fura-2/AM for 30 min at 37 °C. Coverslips with cultured cells were mounted on a perfusion chamber and placed on the stage of an epifluorescence inverted microscope (Nikon Eclipse Ti2) with an image acquisition and analysis system for videomicroscopy (NIS-Elements Imaging Software, version 5.02.00; Nikon). Cells were continuously superfused at room temperature with Hepes-buffered saline containing (in millimolar) 125 NaCl, 5 KCl, 1 MgCl₂, 5 glucose, and 25 Hepes, pH 7.4, supplemented with 0.1% (w/v) BSA. Cells were examined at 40× magnification (Nikon CFI S FLUOR 40× Oil) and alternatively excited with light from a xenon lamp passed through a high-speed monochromator Optoscan ELE 450 (Cairn Research) at 340/380 nm. Fluorescence emission at 510 nm was detected using a cooled digital sCMOS camera PCO Panda 4.2 (Excelitas PCO GmbH) and recorded using NIS-Elements AR software (Nikon). Fluorescence ratio (F340/F380) was calculated pixel by pixel, and the data were presented as ΔF₃₄₀/F₃₈₀. CCh-evoked changes in [Ca²⁺]_i were estimated as the area under the curve measured as the integral of the rise in fura-2 fluorescence ratio 10 min after the addition of the agonist and taking a sample every second.