In vitro magnetothermal stimulation and calcium imaging
Calcium imaging in vitro and fluorescence change quantification were
performed as follows. The TRPV1His-expressing HEK293T
cells were washed three times in PBS and incubated with Fluo-4 (2 µM)
for 30 min at 37 °C. Then, the cells were rewashed with PBS. Next, the
anti-His-FVIOs solution (1 mg/mL) was added to the culture medium of
TRPV1-expressing cells on a dish for 15 min incubation. The unbound
FVIOs in cell culture medium were washed and removed with calcium
imaging buffer (105 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 0.6
mM MgCl2, 10 mM HEPES, 1.2 mM NaHCO3,
100 mM mannitol, and 10 mM glucose) and then imaged in calcium imaging
buffer before and during AMF treatment. A magneTherm system with a
live-cell coil was employed to provide an AMF at 290 kHz and 20 mT.
Calcium fluorescence imaging was performed on a confocal laser
microscope (Nikon) system at 20 × magnification. The fluorescence
intensities (Ft) during AMF exposure were normalized to
the average baseline fluorescence (F0) to calculate the
relative fold change Ft/F0. The
F0 for each examined cell was obtained for the first 20
seconds before AMF treatment. Responsive cells and the standard
deviations of signals were determined during the stimulation period. The
cultured TRPV1His-expressing cortical neurons that
bound with FVIOs were also magnetothermally stimulated for calcium
imaging experiment as the protocol.