Fura 2am

A total of 20 rats, three C57BL/6 mice, and three TRPA1 deficient mice were used for calcium imaging analysis. Twenty-four hours after dissection of the DRG neurons, they were loaded with 4 μM Fura-2AM (Nacalai Tesque, Kyoto, Japan) for 1 h at 37 °C. The coverslip was placed into the recording chamber, and a normal bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid (HEPES), and 10 mM glucose at pH 7.4, adjusted with NaOH, was perfused. Depolarization of the DRG neurons was induced with 50 mM KCl in a normal bath solution. Ratiometric calcium imaging was performed using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742-80-12AG, Hamamatsu Photonics, Hamamatsu, Japan). Dual images (at 340 and 380 nm excitation) were collected every 1 s and analyzed using HCImage Acquisition software (Hamamatsu Photonics, Hamamatsu, Japan). A microscopic field containing 20–30 neurons was randomly selected under the 20X objective.