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Validation of neuronal integrity. (A) Viability assay of iSNs following treatment with increasing concentrations of FdU and AraC for up to 72 h. SDS was used as negative ctrl, 10 μM of FdU for 24 h. The data of n = 3 differentiations, one-way ANOVA with post-hoc Tukey's HSD. * p < 0.05; *** p < 0.001. (B) iSNs generated using our optimized protocol express the sensory neuron marker Nav1.8, the peripheral neuron marker PRPH, and a general neuronal marker TUJ1. The scale bar = 100 μm. (C, D) Spontaneous electrical activity in iSNs was measured at 4 (I), 5 (II), and 6 (III) weeks of maturation using a <t>multi-electrode</t> array system, with activity detectable starting from 4 weeks of maturation. Scale: 50 μV/10 s. (D) Raster plot of one well over 4 (I), 5 (II), and 6 (III) weeks of maturation showing the single and developing network activity. For (C, D) n = 3 triplicates from 1 differentiation. ICC, immunocytochemistry; Nav1.8, voltage-gated sodium channel 1.8; PRPH, peripherin; TUJ1, Beta-III-tubulin.
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Validation of neuronal integrity. (A) Viability assay of iSNs following treatment with increasing concentrations of FdU and AraC for up to 72 h. SDS was used as negative ctrl, 10 μM of FdU for 24 h. The data of n = 3 differentiations, one-way ANOVA with post-hoc Tukey's HSD. * p < 0.05; *** p < 0.001. (B) iSNs generated using our optimized protocol express the sensory neuron marker Nav1.8, the peripheral neuron marker PRPH, and a general neuronal marker TUJ1. The scale bar = 100 μm. (C, D) Spontaneous electrical activity in iSNs was measured at 4 (I), 5 (II), and 6 (III) weeks of maturation using a multi-electrode array system, with activity detectable starting from 4 weeks of maturation. Scale: 50 μV/10 s. (D) Raster plot of one well over 4 (I), 5 (II), and 6 (III) weeks of maturation showing the single and developing network activity. For (C, D) n = 3 triplicates from 1 differentiation. ICC, immunocytochemistry; Nav1.8, voltage-gated sodium channel 1.8; PRPH, peripherin; TUJ1, Beta-III-tubulin.

Journal: Frontiers in Neuroscience

Article Title: Human sensory-like neuron cultivation—An optimized protocol

doi: 10.3389/fnins.2024.1429694

Figure Lengend Snippet: Validation of neuronal integrity. (A) Viability assay of iSNs following treatment with increasing concentrations of FdU and AraC for up to 72 h. SDS was used as negative ctrl, 10 μM of FdU for 24 h. The data of n = 3 differentiations, one-way ANOVA with post-hoc Tukey's HSD. * p < 0.05; *** p < 0.001. (B) iSNs generated using our optimized protocol express the sensory neuron marker Nav1.8, the peripheral neuron marker PRPH, and a general neuronal marker TUJ1. The scale bar = 100 μm. (C, D) Spontaneous electrical activity in iSNs was measured at 4 (I), 5 (II), and 6 (III) weeks of maturation using a multi-electrode array system, with activity detectable starting from 4 weeks of maturation. Scale: 50 μV/10 s. (D) Raster plot of one well over 4 (I), 5 (II), and 6 (III) weeks of maturation showing the single and developing network activity. For (C, D) n = 3 triplicates from 1 differentiation. ICC, immunocytochemistry; Nav1.8, voltage-gated sodium channel 1.8; PRPH, peripherin; TUJ1, Beta-III-tubulin.

Article Snippet: The electrical activity of iSNs was assessed using a multiwell multi-electrode array (MEA) system comprising 24 individual wells with 12 electrodes per well (Multi Channel Systems MCS GmbH, Reutlingen, Germany).

Techniques: Viability Assay, Generated, Marker, Activity Assay, Immunocytochemistry