Functional Calcium Imaging of Mouse DRG Neurons

Functional Ca²⁺ imaging on cultured mouse DRG neurons (2-3 DIV) was performed as described previously (Schnizler et al., 2008 (link)). Neurons on glass coverslips were incubated at room temperature (22°C) for 30 min with 2 μM of the AM form of the Ca²⁺-sensitive dye Fura-2 (Life Technologies). The coverslip was then placed in the recording chamber mounted on the stage of an inverted IX-71 microscope (Olympus Co., Tokyo, Japan) and washed for 10 min before the experiment began. Fluorescence was alternately excited at 340 nm and 380 nm (both 12 nm band pass) using the Polychrome IV monochromator (T.I.L.L. Photonics, Martinsried, Germany), via a 10× or 20× objective [numerical aperture (NA) 0.75; Olympus]. Emitted fluorescence was collected at 510 (80) nm using an IMAGO CCD camera (T.I.L.L. Photonics). Pairs of 340/380 nm images were sampled at 0.2 Hz. Bath application of capsaicin (15 sec) was performed twice with a 5 min interval, and NPs, prostaglandin E₂ (PGE₂), and bradykinin (BK) were applied for 5 min during this interval. The fluorescence ratio (R = F₃₄₀/F₃₈₀) was converted to [Ca²⁺]_i, as described earlier (Schnizler et al., 2008 (link)). Data were processed and analyzed using TILLvisION 4.0.1.2 (T.I.L.L. Photonics) and Origin 7.0 (Microcal, Northhampton, MA) software, and presented as mean ± SEM. All the Ca²⁺ imaging experiments with or without NP/PGE₂/BK treatments were performed in triplicate across three different batches of mouse DRG neuron cultures.