Stable Overexpression of Human TRP Channels in HEK-293 Cells

Human embryonic kidney-293 (HEK-293) cells (ATCC, Rockville, MD) were engineered to stably overexpress human TRPV3 (in pcDNA3.1 D V5/His-TOPO) and other human TRP channels (TRPA1, TRPM8, TRPV1, TRPV2, and TRPV4) as previously described (Deering-Rice et al. 2011 (link)). HEK-293 cells were grown in DMEM:F12 (Invitrogen, Carlsbad, CA) containing 5% fetal bovine serum (FBS) and 1× penicillin/streptomycin (Invitrogen). HEK-293 cells stably overexpressing the various TRP channels were maintained in DMEM:F12 supplemented with 5% FBS, 1× penicillin/streptomycin, and 350 μg/mL Geneticin (Invitrogen). Cells were subcultured using trypsin and plated into 1% gelatin-coated 96- or 384-well plates for calcium imaging experiments. Human immortalized keratinocytes, HaCaT cells, were provided by Dr. Douglas Grossman, M.D., Ph.D., of the Department of Dermatology, University of Utah. HaCaT cells were cultured in DMEM containing 5% FBS and 1× penicillin/streptomycin/glutamine. Cells were subcultured using trypsin and plated into 96-well plates for experiments.