and allowed to cool at room temperature. dsRNA concentration was measured by UV spectroscopy [65]. Neurosphere cultures maintained in proliferation medium for 7 days were treated with 50 mg/ml of PIC for the last 24 hours of the culture period. We observed robust phenotypic changes in neurosphere cells that were treated with 50 mg/ml of PIC without inducing significant loss of viability; hence, all phenotypic testing was performed with cultures treated with this dose of poly I:C.. Antibodies Primary antibodies included: mouse anti-BrdU (1:50, Clone: B44, Becton Dickinson, San Jose, CA), mouse anti-PAX6 (1:100, developed by Atsushi Kawakami and obtained from Development Studies Hybridoma Bank, University of Iowa, Iowa city, IA), mouse anti-TBR2-PE (1:100, Clone: 21 Mgs8, eBioscience, San Diego, CA), chicken anti-mouse serum TLR3 (1:500, Sigma- Aldrich, St. Louis, MO), rat serum anti-mouse TLR3 (1:500, eBioscience, San Diego, CA), mouse anti-Nestin-Alexa Fluor 647 (1:20, Clone: 25/Nestin, BD Pharmingen, San Jose, CA), mouse anti-Tubulin-b-III-NorthernLights 637 (1:20, Clone: TuJ-1, R&D Systems, Minneapolis, MN), mouse anti-GFAP-Alexa Fluor 488 (1:50, Clone: 131-15019, Invitrogen, Carlsbad, CA), mouse anti- oligodendrocyte marker O4-PE (10ml, Clone: O4, R&D Systems), goat anti-mouse Patched (1:20, R&D Systems), Rat anti-Gli1- FITC (1:100, R&D Systems), rabbit anti-mouse Phospho-MEK1/ 2 (1:100, Cell Signaling Technology, Danvers, MA), rabbit anti- mouse Phospho-ERK (1:100, Cell Signaling Technology), and hamster anti-mouse CD95-FITC (1:100, Clone: Jo2, BD Phar- mingen). Secondary antibodies included donkey anti-mouse-APC, donkey anti-goat-PE, donkey anti-mouse-FITC, donkey anti rat- FITC, (1:1000, Jackson Immunoresearch, West Grove, MA), goat anti-rat-PE (1:100, R&D Systems), and goat anti-rabbit-APC (1:100, R&D Systems). Flow cytometry isotype controls were PE rat IgG2a k, FITC rat IgG2a k, APC mouse IgG2a k, FITC mouse IgG1 k, and APC mouse IgG1 k isotype (BD Biosciences, Bedford, MA). Flow cytometry BrdU was added to cortical neurosphere cultures at 10 mM, 12 hours prior to culture processing. Neurospheres were pelleted by centrifugation at 800 xg for 5 minutes. Cell pellets were then suspended in 2 ml of trypLE stable trypsin replacement enzyme (Invitrogen, San Diego, CA) and incubated at 37uC for 15 minutes. The neurosphere suspensions were pelleted again and resuspended in 2 ml of NeuroCult NSC proliferation media supplemented with 250 U/ml of DNAse I (Sigma-Aldrich, Saint Louis, MO, USA). Single cell suspensions were obtained by trituration with fire-polished glass Pasteur pipettes. Intracellular BrdU was measured using the BrdU Flow Kit (BD Biosciences, Bedford, MA). Cells were resuspended in 50 ml of staining buffer (PBS with 1.0% FBS). Cells were fixed, permeabilized, and treated with DNAse (30 mg per tube) to expose incorporated BrdU. Cells were resuspended in 50 ml of BD Perm/fix buffer containing anti- BrdU-FITC antibodies (1:50) and relevant antibodies specific for detection of intracellular antigens, followed by staining with secondary antibodies. Cells were washed and resuspended in staining buffer and analyzed by multicolor flow cytometry on a LSRII Analyzer (Becton Dickinson, Franklin Lakes, NJ). After gating to exclude dead cells and debris based on forward and side scatter, data were analyzed using FACS DiVa acquisition software (Becton Dickinson, Franklin Lakes, NJ) and FlowJo6.1 (Tree Star, Ashland, OR). TLR3 Regulates Neural Stem Cell Proliferation Apoptosis assays Cell suspensions of cortical neurospheres were washed twice with PBS, resuspended in 0.1 ml Annexin V binding buffer (BD Biosciences), and incubated with 5 ml of FITC-conjugated Annexin V (BD Biosciences) and 10 ml of propidium iodide (PI) for 15 min at room temperature. Cells were immediately analyzed by flow cytometry on a FACSCalibur (Becton Dickinson). Data were obtained using CellQuest acquisition software (Becton Dickinson) and analyzed using FlowJo6.1 (Tree Star, Ashland, OR, USA). Cells stained with Annexin V-FITC alone and PI alone were used as controls. Statistical analysis StatView version 5.0.1 software (Windows version; SAS Institute, Cary, NC, USA) was used for all statistical analyses. Mann–Whitney U-tests (independent variable: dose group) were used for group comparisons requiring nonparametric analytic approaches. For all tests, statistical significance was assumed where p,0.05. Supporting Information Figure S1 Phase contrast images of primary neuro- sphere cultures. EGF-responsive murine cortical cells, isolated from GD 14 WT embryo, were grown for 1 day or for 7 days in proliferation medium supplemented with 20 ng/ml of EGF. (a) Small clusters of cells were visible one day after plating. (b) Spherical, bright phase and viable neurospheres were identified after 7 days in vitro. Magnification, 10X. (TIF) Figure S2 Percentage of nestin-positive cells at different passages. Cortical cells were obtained from WT embryos at GD14 and cultured in EGF-containing proliferation medium (20 ng/ml) for 7 days to form neurospheres. Cells were passaged once a week by enzymatic and mechanical re-dissociation and re- plated. (a) Representative histograms showing the percentages of nestin+cells in primary and (b) secondary neurospheres (2nd passage) that were in culture for 7 days. Numbers in gates represent the percentages of nestin+ cells. Three independent cell culture assays were performed with cells isolated from embryos (n = 12); data from one representative assay is shown. (TIF) Figure S3 In vitro differentiation of WT neurospheres. Dissociated cells from WT primary cortical neurospheres were plated onto poly-D-lysine/laminin-coated six-well plates (Sigma) and differentiated in serum-free DMEM/F-12 medium (Invitro- gen) supplemented with B27 (Invitrogen), Neurocult NSC supplement (Stem cell) and 1% Fetal calf serum (Invitrogen) for 7 days. (a–c) During differentiation, neurosphere-derived cells lost their spherical shape and flattened to form a monolayer (bright field, a). Immunofluorescence analysis showed that WT neuro- spheres were multipotent and expressed genes such as bIII-tubulin (red, b) or GFAP (red, c), markers characteristic for neurons and astrocytes, respectively. WT neurospheres also expressed nestin (green, b, c). Nuclear staining (blue, b, c). (d–f) Representative histograms showing the percentages of differentiated WT neuro- sphere-derived cells expressing tubulin-b-III (d), O4 (e), and GFAP (f). Numbers in gates represent the percentages of each sub- population. Overlays represent the mean fluorescence intensity expressed as a percentage of the maximum expression: ——, cell lineage marker; ——, isotype control. (TIF) PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e26766