Calcium Imaging
Intracellular calcium imaging experiments were performed as previously
described (Dembla, Behrendt et al. 2017). Briefly, cultured rat sensory
neurons (DRGs) were incubated at room temperature with 5 μM Fura 2‑AM
(1 mM stock in DMSO + 0.02% pluronic F-127) for 30 min in growth
medium. Following the application of Fura 2‑AM, cover‑slips were
transferred to a closed recording chamber (Warner Instruments) and
continuously perfused with the extracellular solution for 5 min.
Fluorescence was monitored every 2 seconds and images were taken with
the DS-Qi1 (Nikon, Japan) at 510 nm wavelength. Alternate excitation at
340 and 380 nm wavelengths were filmed using a Polychrome V
monochromator, mounted on a Nikon TE2000 inverted microscope (with 20x
Sfluor objective; N.A. 0.5). After a baseline period of ca. 2 minutes,
ligands (as shown in figures) were superfused onto the cells, using a
gravity-driven perfusion system, which was controlled by ALA VC3‑8 valve
control system. Fluorescence intensities of ratio images (340/380 nm) on
regions of interest (ROI) (including the whole cell) were quantified by
NIKON NIS-Elements software after subtraction of background. To
distinguish between neuronal and non-neuronal cells, a high potassium
(high K+) solution was used to depolarize the cells at
the end of the superfusion protocol.