Functional characterization of HC and SCD iSNs using traditional and high-throughput calcium assays. HC and SCD iSNs respond to 50 mM KCl, 100 μM glutamate, and 50 μM ab-meATP in traditional calcium imaging setup (panels A and B show representative traces). (C) Concentration curve shows that HC and SCD iSNs have few calcium responders to 0.1 and 1 μM capsaicin (subthreshold), identifying 10 μM capsaicin as appropriate to capture iSN response consistent with previous studies (panel D shows representative traces). (E) Schematic of Fluo4-NW calcium flux assay adapted to capture population responses of entire wells of iSNs to allow for high-throughput investigation of iSN sensitization. (F) Concentration curve using population-based Fluo-4NW assay allows for increased capture of subthreshold (0.1 μM, blue; 1 μM, pink) capsaicin although with low magnitude responses (F, right). Low-concentration (10 μM, teal) and high-concentration (50 μM, purple) capsaicin agonism resulted in consistent and high calcium response rates with increased magnitude (1-way ANOVA, ∗P < .05, ∗∗∗P < .0005). Consistent with calcium imaging data, Fluo-4NW assay shows no significant differences between HC and SCD iSN response to 50 mM KCl (G), 100 μM glutamate (H), and 50 μM ab-meATP (I), 10 μM capsaicin (J), or 50 μM capsaicin at baseline (K) (Student t tests; ns, not significant). Points in the violin plots represent individual wells of iSNs, and means are represented as a dashed line.