Cell culture and transfection
The human embryonic kidney cells (HEK293) were cultured in the Dulbecco’s minimal essential medium with supplement of 10 % of fetal bovine serum (FBS) at 37℃ with 5 % CO2. HEK293 cells were seeded in a 6-well plate for intracellular calcium measurement. For whole-cell patch clamp recordings, cells were cultured on glass coverslips. The HEK293 cells were transiently cotransfected in 350-mm petri dishes or a 350-mm well on a 6-well plate with 1600 ng of cDNA plasmid encoding wild-type or mutant human TRPA1 and and other TRP channels, using Lipofectamine 2000 (Invitrogen). The cells were used after 24-48 h transfection. Human TRPA1 plasmids (Gene ID: 8989) including wild-type or mutants were verified by DNA sequencing.
Measurement of intracellular calcium in fluorescent imaging plate reader FlexStation 3 assay
The Cal-520TM PBX Calcium Assay Kit (AAT Bioquest, Sunnyvale, CA) was used to detect the change of intracellular calcium in a population of cells with Multi-Mode Microplate Reader in FlexStation 3 assay (Molecular Devices, San Jose, CA). HEK293 cells were transiently transfected with cDNA of TRP channels before seeded at a density of ~40,000 cells per well in a 96-well black-walled plate (Thermo Fisher Scientific, Waltham, MA) for an overnight culture at 37℃ with 5 % CO2. Cells were incubated with the calcium fluorescent dye provided in the Cal-520TM PBX Calcium Assay Kit at 37℃ for 1.5 h, and the values of relative fluorescence unit (RFU) were measured by FlexStation3 under the wavelength at 485 nm (excitation) and 515 nm (emission) with an interval of 1.6 s (Sun et al., 2017).