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.