Embryonic hippocampal cultures were prepared from embryonic day 17–18 CD1 mice following the published protocol [59 (link)] with modifications. All procedures were performed in accordance with animal welfare guidelines at the University of Toronto and were approved by the institutional animal care and use committee.
Dissected hippocampi were digested with 0.025% Trypsin/EDTA at 37 °C in HBSS (Hanks balanced salt solution; Sigma, St. Louis, MO, U.S.A) for 15 min and washed with pre-warmed medium to stop digestion. Cells were triturated approximately 10 times with a 1000 μL tip. Neurons were centrifuged at 1000 rpm for 5 min, supernatant was removed and cells were re-suspended in culture media. Cell density was determined using an Improved Neubauer hemocytometer and low-density cultures were plated on Poly-D-Lysine (0.1 mg/ml Sigma) coated glass coverslips (18 mm #1.5, Warner Instruments) at 25,000 neurons/cm2. Hippocampal neurons were cultured using a spatially separated ring of cortical neurons for neurotrophic support following the method published previously [60 (link)]. Neurons were plated in Neurobasal medium (Invitrogen, Carlsbad, CA, U.S.A.) with 2% B27 (Invitrogen), 1 × Pen/Strep (Gibco), and 2 mM GlutaMAX (Invitrogen) at 37C with 5% CO2.
Dissected hippocampi were digested with 0.025% Trypsin/EDTA at 37 °C in HBSS (Hanks balanced salt solution; Sigma, St. Louis, MO, U.S.A) for 15 min and washed with pre-warmed medium to stop digestion. Cells were triturated approximately 10 times with a 1000 μL tip. Neurons were centrifuged at 1000 rpm for 5 min, supernatant was removed and cells were re-suspended in culture media. Cell density was determined using an Improved Neubauer hemocytometer and low-density cultures were plated on Poly-D-Lysine (0.1 mg/ml Sigma) coated glass coverslips (18 mm #1.5, Warner Instruments) at 25,000 neurons/cm2. Hippocampal neurons were cultured using a spatially separated ring of cortical neurons for neurotrophic support following the method published previously [60 (link)]. Neurons were plated in Neurobasal medium (Invitrogen, Carlsbad, CA, U.S.A.) with 2% B27 (Invitrogen), 1 × Pen/Strep (Gibco), and 2 mM GlutaMAX (Invitrogen) at 37C with 5% CO2.
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