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. 2017 Aug 16;7:8486. doi: 10.1038/s41598-017-08459-z

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© The Author(s) 2017

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Cell viability does not differ by sex in hippocampal and cortical cultures. Sex-specific neuron-glia co-cultures were established from P0 mouse hippocampi (a,b) or neocortices (c,d). On DIV 9, cell viability was analyzed by measuring lactate dehydrogenase (LDH) release into the medium (a,c) or by quantifying the percentage of live cells in cultures stained with calcein AM (biomarker of live cells) and propidium iodide (biomarker of dead cells) using Metamorph image analysis software (b,d). As a positive control, a subset of cultures for each experimental condition were lysed with 0.2% Tritox X (Lysis). In box plots, “+” indicates the mean; whiskers, the 10–90^th percentile (n = 6–9 wells per sex from at least three independent dissections). Live dead cell analysis was conducted on imaged sites (9 fields/well) which contained greater than 250 total cells counted per field. Significant differences were determined using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis for parametric data (b) or Kruskal Wallis test followed by Dunn’s post hoc analysis for nonparametric data (a,c,d). Asterisk indicates a significant difference between groups at p ≤ 0.05.