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. 2023 Sep 22;14:5898. doi: 10.1038/s41467-023-41603-0

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Experimental setup for motor neuron (MN)-microglia (MG) co-culture. b Percentage of MNs/MG in co-cultures of healthy control (HC) MNs and HC, isogenic control (IC), and C9orf72 mutant (C9) MG showing a ~ 1:1 ratio (n = 3 microglial lines (C9-HC comparison), n = 1 microglial line (IC-C9-2 comparison), n = 3 differentiations). c Left: Exemplar images showing the apoptosis marker cleaved caspase 3 (CC3) in HC MNs in co-culture with MG. Scale bars: 15 μm. Right: Quantification of relative fold change of CC3 expression (n = 3 microglia lines (C9-HC comparison), n = 1 microglial line (IC-C9-2 comparison), n = 3 differentiations). d, e Patch clamping analysis of resting membrane potential and capacitance of HC MNs in co-culture with MG (datapoints are single MNs from co-cultures with n = 3 microglial lines (d, +HC: n = 18, +C9: n = 17; e, both n = 17), n = 1 differentiation). f Multi-electrode array analysis (MEA) of mean firing rate of HC MNs in co-culture with MG (n = 3 microglial lines (C9-HC comparison), n = 1 microglial line (IC-C9-2 comparison), n = 2 wells per n = 3 differentiations). g Left: exemplar images of neurites of HC MNs in co-culture with MG. Right: quantification of neurite outgrowth (n = 3 microglial lines (C9-HC comparison), n = 1 microglial line (IC-C9-2 comparison), n = 3 differentiations). Scale bars: 50 μm. h Left: exemplar images of synaptophysin (sy-physin) staining in co-culture of HC MNs with MG. Right: quantification of the number of synaptophysin particles (n = 3 microglial lines (C9-HC comparison), n = 1 microglial line (IC-C9-2 comparison), n = 3 differentiations). Scale bars: 50 μm. i Relative fold change in co-culture supernatant for selected targets using a cytokine array (pooled from co-cultures of HC MNs with n = 3 microglial lines (C9-HC comparison), n = 1 microglial line (IC-C9-2 comparison), n = 3 differentiations). j ELISA quantification of DPP4 in co-culture supernatants from HC MNs and MG (n = 3 microglial lines for C9-HC comparison, n = 1 microglial line for IC-C9-2 comparison, n = 3 differentiations). Single data points and means ± SEM. **P < 0.01; ***P < 0.001; ns not significant. Two-tailed unpaired t-test (c–e, g, h, j), two-way ANOVA with Tukey’s post-hoc test (f). The graphics for panel a were created using Biorender.com. Source data are provided as a Source Data file.