Fura 2 am

A minimum of 1 × 10⁵ AM were plated in a 35 mm dish and incubated for 2 h. The cells were prepared by incubation for 30 min in 1 ml loading buffer containing Ringer solution (140 mM NaCl, 5 mM KCl, 5 mM CaCl₂, 2.5 mM MgCl₂, 1 mM HEPES) and 7.5 μM Fura-2 AM (Enzo, Farmingdale, USA, cat. ENZ-52006) like described before (Gelis et al. 2016 (link)). After removal of the extracellular Fura-2 AM by washing with Ringer solution, Ca²⁺-ratiometric imaging was performed using a light source (EL6000, Leica), a Leica inverted confocal microscope (DMI6000 CS, Leica) with a 20× objective (UPLSAPO, Olympus, Tokyo, Japan). The images were recorded at 1 Hz by the DFC360 FX (Leica, Wetzlar, Germany), and the integrated fluorescence (f 340 nm/f 380 nm) of each cell was measured using the Leica Application Suite Advanced Fluorescence (LAS AF). The odorants were pre-diluted in DMSO and then adjusted to the final concentration in Ringer solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCL₂, and 10 mM Hepes). The inhibitors MDL-12,330A (10 µM; Sigma-Aldrich, St. Louis, USA, cat: M182) and Oxyphenylon (300 µM; Henkel) were also pre-diluted in DMSO. EGTA (10 mM; Sigma-Aldrich, St. Louis, USA, cat: 03777) was prepared in water.

Enhanced NaTrium Green-2 AM (ENG; Shanxi, China), N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide (MQAE; Beyotime, China), and calcium-sensitive dye Fura-2 AM (Molecular Probes, Abcam, Cambridge, MA, USA) were used to detect intracellular sodium, chloride, and calcium ion concentrations, respectively. A549 cells were incubated with 5 μM MQAE for 30 min at 37 °C and washed 5 times with Krebs-HEPES buffer, while cells were incubated with 5 μM Fura-2 AM for 30 min at 37 °C, and then treated with Hanks’ balanced salt solution (HBSS) and incubated for another 30 min. A549 cells were incubated with 4 μM ENG-AM for 30 min at 37 °C, then treated with DMEM containing 1% FBS and incubated for another 30 min. The fluorescence intensity was detected using fluorescent microscopy (Thunder; Leica), and fluorescence images were obtained every 60 s. The normalized value of ENG fluorescence intensity (Ft/F0), MQAE fluorescence intensity (F0/Ft), and Fura-2 AM fluorescence intensity (Ft/F0) was calculated based on the ion fluorescence just after (Ft) or before (F0) the application of stimulation for 15 min, respectively.