Calcium Imaging with primary sensory neurons

Primary sensory neurons were cultured as described previously (Sisignano et al., 2012). For calcium imaging experiments, neurons were stained with Fura-2-AM (Thermo Fisher) for at least 60 min at 37 °C and washed afterwards twice with Ringer’s solution consisting of 145 mM NaCl, 1.25 mM CaCl₂ ×2H₂ O, 1 mM MgCl₂ x6 H₂O, 5 mM KCl, 10 mM D-glucose, and 10 mM HEPES adjusted to a pH of 7.3. To investigate the effect of SA1P or LPC 24:0 on different TRP channels, sensory neurons were incubated with the lipids for 1 min at a concentration of 1 or 10 µM, respectively. The gold standard agonists for TRPV1 and TRPA1 were capsaicin (200 nM, 20 s) and AITC (allyl isothiocyanate, 75 µM, 30 s). Fingolimod was used at a concentration of 1 µM and pre-incubated for 1 hr prior to measurement. As a positive control, final stimulation with KCl (50 mM, 1 min) was used to depolarize all neurons. All stimulating compounds were dissolved in Ringer’s solution to their final concentrations.

The calcium imaging data were analyzed using descriptive statistics. All calcium imaging data are presented as the mean ± SEM. Normal distribution was confirmed using the Shapiro-Wilk test. For experiments comparing only two groups, unpaired and heteroscedastic Student’s t-tests were conducted following Welch’s correction. When comparing more than two groups, one-way analysis of variance (ANOVA) was used, and for the comparison of more than three groups, two-way ANOVA was conducted. For all statistical analyses of the calcium imaging data, the software GraphPad Prism was used (version 9.5, GraphPad Software, Boston, MA, USA). Statistical significance was set at p-value <0.05.