Calcium imaging

The day before the experiment cells were seeded to reach the density of 25′000 cells per well in sterile 96-well assay plates (Corning Life Sciences) using a Ham’s F12 medium supplemented with 10% Fetal Bovine Serum, 100 units/ml Penicillin–Streptomycin and 1 μγ/ml Tetracycline Hydrochloride (Sigma Aldrich) to allow the expression of the human TRPA1. For the control (CHO background cells without TRP channels) and the CHO_hTRPV1, cells were seeded in the same conditions but without Tetracycline. Plates were let 18–24 h in a humidified 5% CO2 incubator at 37 °C. The day of the experiment cells were loaded with a 2 mM Fura-2AM (Life Technologies) and 0.04% Pluronic acid F-127 (Life Technologies) solution and incubated 45 min in a humidified 5% CO2 incubator at 37 °C. Then the cells were washed with calcium buffer (137 mM NaCl, 4 mM KCl, 1.8 mM CaCl₂, 1 mM MgSO₄, 10 mM Hepes, 10 mM (D) Glucose, buffered at pH 7.4) and let 15 min in a humidified 5% CO2 incubator at 37 °C. The 96-well plates were then placed into the plate reader (FLEXstation; Molecular Devices) which added the compounds previously diluted in the calcium buffer from a source plate to the cell plate and measured changes in Ca^2+-induced fluorescence intensity (FI) of dye at F340/380 (_ex1_340 nm;_ex2_380nm; _em _ 510 nm).

The fluorescence intensities (FI) were normalized as described below:

FI_max being the fluorescence of agonists used as positive controls at a concentration giving maximum activation (data not shown):

Single cell calcium imaging experiments were performed on an AXIO Observer. D1 (Zeiss), a digital camera ORCA-Flash4.0 (Hamamatsu), a High Speed Polychromator System VisiChrome and acquired by VisiView software (Visitron Systems GmbH). The day of experiment, DRG were loaded during 45 min in Fluo 4 containing probenecid (Fluo-4 NW Calcium Assay Kit, Molecular Probes, cat.number {"type":"entrez-nucleotide","attrs":{"text":"F36206","term_id":"4821831","term_text":"F36206"}}F36206). Coverslips were mounted in a bath-imaging chamber RC-25F, with VC-8 Valves Controller through a dual automatic temperature controller TC-344B (Warner Instruments). DRG are continuous perfused at 37 °C with HBSS, 20 mM HEPES, 2 mM CaCl2, pH7.4 (Sigma). DRG were first perfused 10 min for washing, and stimulations were performed during 50 s with 25 mM Tiglic Aldehyde, 5 mM p-Anisaldehyde, 3 mM Cuminaldehyde, 100 µM Cinnamaldehyde (Sigma) and 10 µM Capsaicin (Sigma). 50 mM KCl (Sigma) was used as the positive control. Fluorescence was measured using excitation at 494 nm and emission at 516 nm.