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.