Primary rat cortical cultures were prepared from embryonic day 18 Sprague-Dawley rat embryos (Charles River Laboratories, Seattle, WA). Brains were isolated, and dissected cortices were incubated for 40 min in DMEM +0.027% trypsin as described previously (Wilcox et al. 1994 (link)). Cells were then washed in saline, triturated, resuspended in neurobasal media supplemented with B27, and plated on poly-L-lysine-coated 6-well (9.4-cm2 growth area) or 24-well (1.9-cm2 growth area) plates (USA Scientific, Ocala, FL) at a concentration of 500,000 cells/ml. Cultures contained approximately 90% neurons and 10% astrocytes/glia and were maintained in neurobasal media supplemented with B27 at 37 °C with 5% CO2 as described previously (Gannon et al. 2017 (link); Akay et al. 2011 (link)). On 10 days in vitro (DIV), 20% fresh media was added. Cells were treated on DIV 14–16.
Sprague dawley rat embryos
Manufactured by Charles River Laboratories
Sourced in Japan
Sprague-Dawley rat embryos are a widely used model organism in biomedical research. They are a specific strain of laboratory rat commonly employed for studies in areas such as developmental biology, teratology, and toxicology.
Automatically generated - may contain errors
Lab products found in correlation
Gentamicin, by Thermo Fisher Scientific (1 mentions)
B 27 plus supplement, by Thermo Fisher Scientific (1 mentions)
Neurobasal plus medium, by Thermo Fisher Scientific (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Papain dissociation system, by Worthington (1 mentions)
Poly l lysine (pll), by AGC Techno Glass (1 mentions)
2 protocols using sprague dawley rat embryos
1
Primary Rat Cortical Cultures: Preparation and Maintenance
2
Culturing Cortical Neurons for NMDA Assays
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Cortical neurons were prepared from 16-day-old Sprague Dawley rat embryos (Charles River Laboratories Japan, Inc., Yokohama, Japan). Cortices were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation, Lakewood, NJ, USA) according to the manufacturer’s instructions, and the cells were seeded onto 96-well plates pre-coated with poly-L-lysine (AGC Techno Glass Co., Ltd., Shizuoka, Japan) at a cell density of 5.0 × 104 cells/well. The cells were placed in a 5% CO2 incubator at 37°C and cultured for 20 days in the Neurobasal Plus Medium supplemented with 2% B27 Plus Supplement and 40 μg/mL gentamicin (all from Thermo Fisher Scientific Inc., Waltham, MA, USA). The culture medium was exchanged every 3–4 days and removed just before the application of NMDA. The cells were simultaneously incubated for 2 h with the test compounds and NMDA, and the cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays in accordance with the manufacturer’s instructions (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega Inc., Madison, WI, USA).
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