Black microtiter plate

To measure intracellular IP₃ the HitHunter^® Inositol (1,4,5) Triphosphate Assay (DiscoveRx, USA) was used according to manufacturer′s protocol. HEK293 cells stably transfected with hP2RY6 were seeded in a 384-well Black microtiter plate (15,000 cells/well) (Greiner Bio One, Germany) and incubated with indicated concentrations of UDP and PGE₂-G for 20 seconds. Reaction was stopped by adding 5 µl 0.2 N perchloric acid and measurement of IP₃ was performed with the Fusion AlphaScreen multilabel reader (PerkinElmer Life Sciences) according to manufacturer′s protocol. To measure IP₁, HEK293 cells expressing wildtype and mutant P2Y₆ were seeded into 384-well plates (5,000 cells/well) 24 h prior assay. After aspiration of the medium, cells were incubated with indicated concentrations of agonists/antagonist for 1 h. IP₁ measurements using the IP-one HTFR^® assay kit (Cisbio assays, USA) were performed according to manufacturer′s protocol. The assays were performed with a final concentration of 1% DMSO.

During the experiments, the maximum Fv/Fm (maximum photochemical yield) was measured using a PAM 2500 fluorometer, as described in [36 (link)]. ROS levels in G. emersonii cells were detected using an ROS assay kit (S0033S, Beyotime, China). Briefly, 5 mL of mixed cell culture was centrifuged at 5000× g for 5 min, and the cell pellets were resuspended in 1 mL basal medium containing 10 μM DCFH-DA. The suspension was incubated in the dark at 37 °C for 30 min. After labeling, the cells were washed three times with basal medium and resuspended in the original culture medium. Subsequently, the cell suspension was transferred to each well of a black microtiter plate (Greiner Bio, Chimney well, Germany). Fluorescence was analyzed using a fluorescence microplate reader (TECAN, M200 PRO) with excitation and emission wavelengths of 488 and 525 nm, respectively. For each treatment and control, three replicates were performed.

BON and LNCaP cells were seeded onto a 96-well culture plate (black microtiter plate, Greiner Bio-one), respectively at a density of 10⁵ and 0.5×10⁵ cells per well. 24 hours later, cells were loaded with 2.5 µM of fluo-4 acetoxymethyl ester (Molecular Probes), as previously described [18] (link). Calcium imaging was performed using an inverted epifluorescence microscope (CK40 Olympus) equipped with a digital camera (ORCA-ER, Hamamatsu Photonics). Ca²⁺ reponses were observed at 460–490 nm excitation and ≥ 515 nm emission wavelengths. Data acquisition and analysis was performed using the SimplePCI software (Hamamatsu, Compix). Odorants and mineral oil were prepared extemporaneously by a first dilution into DMSO and then serial dilutions into Hanks’ salt solution (Eurobio) supplemented with 20 mM Hepes, pH 7.2. Stimuli were tested at concentrations that do not elicit calcium responses in mock-transfected cells. 1 µM isoproterenol (Sigma-Aldrich) was applied as a positive control. The Ca²⁺ signal was measured as the relative change in fluorescence intensity ΔF/F =  (F–F₀)/F₀, where F₀ is the fluorescence level before stimulation. Results were expressed as the mean of the ΔF/F of at least twenty cells.