Mustard oil

Calcium imaging was performed as previously described.³⁹ (link) Cultured DRGs were imaged by using a 10× objective on an Olympus IX51 microscope controlled by cellSens software (Olympus Canada, Toronto, ON, Canada). Cultured DRG neurons were loaded in HBSS (Gibco) with 2 μmol/L Fura-2AM (Life Technologies, Carlsbad, CA) for 30 minutes at 37°C and then washed with HBSS for 20 minutes at 37°C. The recording chamber was continually perfused at 37°C with external solution (in mmol/L: 140 NaCl, 5 KCl, 10 HEPES, 10 glucose, 1 MgCl₂, 2 CaCl2; pH adjusted to 7.3–7.4 with 10N NaOH). To examine TRPV1 or TRPA1 activity, cells were perfused with doses of the TRPV1 agonist capsaicin (100 nmol/L and 1 μmol/L; Sigma) or the TRPA1 agonist mustard oil (100 nmol/L and 1 μmol/L; Sigma) diluted in external solution at a rate of ∼1 mL/min. Fluorescence was measured during excitation at 340 and 380 nm, and the ratio of the fluorescence emission at 510 nm was monitored. The baseline was monitored for 120 seconds before perfusion with agonist. Images were acquired every 1 second and processed by using ImageJ (NIH, Bethesda, MD) by drawing discrete regions of interest around cells that responded to KCl (70 mmol/L). Data are expressed as a fold change from baseline fluorescence normalized to background fluorescence (ΔF/F0) on raw traces and were analyzed by using area under the curve.