Cell culture and transfection
HEK293T cell culture: Before in vitro experiments, the cells were authenticated and checked for mycoplasma contamination. Human embryonic kidney (HEK293T) cells were plated sparsely on culture dishes and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. For calcium imaging, the cells were carefully transfected with a certain number of plasmids encoding TRPV1 using 1 μL of a standard transfection reagent (GenEscortTM III) with 2 μg of total DNA. Magnetothermal stimulation was performed one day after transfection of the genetically encoded 293T cells.
Cortical neural cell culture: The whole brain was transferred to PBS containing glucose (33 mM), penicillin-streptomycin (1%, v/v) and washed. The cortical tissues were dissected in a trypsin solution (0.25%, 37 °C , 30 min), which was quenched using horse serum (10%, Fisher Scientific) in neurobasal medium. The dissociated tissues were triturated and filtered. The obtained cells were plated onto poly-L-lysine-coated culture dishes. Unattached cells were removed away after 2 h of incubation. The attached cells were cultured in neurobasal medium containing B-27, and half of the medium was replaced every 2 days. Five-day-old cortical neural cells were transfected by adding 2 μL of Lenti-TRPV1-p2A-mCherry (1×1012 transducing units/mL). After a 5-day induction period, magnetothermal stimulation was performed for calcium imaging of the TRPV1-expressing cortical neural cells.