pre-warmed medium was added. Medium consisted of Dulbecco's Modified Eagle's Medium with Nutrient Mixture (Ham's) F-12 medium (DMEM/F12) with glutamine (Invitrogen; 11320-082) supplemented with N2 (Invitrogen; 17502-048), 20 ng/mL FGF-2 (Peprotech; 100-18B), 20 ng/mL EGF (Peprotech; AF-100-15), and 1% antibiotic-antimycotic (Invitrogen; 15240062). The tissue was mechanically dissociated and cells were filtered through a 70-μm cell strainer (VWR; 21008-952), plated on poly-D-lysine coated 6-well plates (50 μg/mL poly-D-lysine per plate; Sigma-Aldrich; P0899), and adherent monolayer proliferating cultures were established and maintained under proliferating conditions using complete medium with growth factors at 37 °C and 5% CO2. One cell culture plate consisted of 8–9 embryos from one dam; at least 6 different dams for each exposure and each culture condition were used for array assessment (n = 6). Differentiated NPC cultures were derived by removing growth factors (FGF-2, EGF) from the medium after 3 passages or 10 days in vitro (DIV). NPCs were allowed to differentiate up to 10 days with up to 2 passages if necessary (Roitbak, Thomas, Martin, Allan, & Cunningham, 2011).
