TLR3 Regulates Neural Stem Cell Proliferation Figure 1. Phenotypic analysis of EGF-responsive primary cortical neurospheres. Primary neurospheres were obtained from cerebral cortex cells isolated from WT and TLR32/2 embryos at GD14. Cell populations in neurospheres were assayed by using flow cytometry. (a) Percentages of nestin-, GFAP- and Tubulin-b-III-expressing cells in neurospheres cultured for 24 hours or 7 days in proliferation medium supplemented with 20 ng/ ml of EGF. Three independent experiments were performed with neurosphere-derived cells isolated from WT and TLR32/2 embryos (n = 12). Results are expressed as percentages of total gated cells. Values represent mean 6 SEM. (b) Dot plots of PAX6, TBR2, and nestin expression in cells derived from WT and TLR32/2 cortical neurospheres cultured for 7 days. Numbers in quadrants represent the percentages of each subpopulation. Three independent cell culture assays were performed with cells isolated from WT and TLR32/2 embryos (n = 12); data from one representative assay is shown. (c) Dot plots of TLR3 expression in nestin+, PAX6+ and TBR2+ WT cortical neurosphere cells cultured for 7 days. Numbers in quadrants represent the percentages of each subpopulation. Three independent cell culture assays were performed with cells isolated from WT embryos (n = 12); data from one representative assay is shown. doi:10.1371/journal.pone.0026766.g001 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26766