Neurobasal media

Hippocampal neuronal cultures were prepared from 3- to 6-day-old Sprague-Dawley rats. All procedures used in this study for handling and sacrificing the animals were in strict compliance with the guidelines of Korean animal protection law and approved by the Institutional Animal Care and Use Committee of Ewha Womans University School of Medicine (Permit Number: 13-0220). Briefly, hippocampi were dissected from 3- to 6-day-old rats into DMEM (Biowest), Trypsinized for 1 hour at 37°C with 0.25% Trypsin/0.53 mM EDTA (Gibco/BRL), triturated with fire-polished Pasteur pipettes, and plated in 6-well plates coated with poly-L-lysine (Sigma-Aldrich) at a density of 4×10⁵ cells/well in Neurobasal media (Gibco/BRL) with 10% FBS. After 16 h, the media were changed to Neurobasal media supplemented with serum-free supplements containing 0.5 mM L-glutamine, 2% B-27, 1% N-2, and 1% penicillin/streptomycin (Invitrogen) for further culturing. Half of the media were changed every 2 days by aspirating and replacing it with fresh culture media for 14 days, at which time Aβ_1–42 treatment was initiated.

Primary hippocampal neurons are isolated from E17 Sprague Dawley rat pups (Charles River, Wilmington, MA, USA) following the specific regulations approved by the Johns Hopkins Institutional Animal Care and Use Committee (protocol RA12M213). Dissociated neurons are loaded into the somal compartment at a density of 25x10⁶ cells/mL in a mixture of neurobasal media (Gibco Life Technologies; Grand Island, NY, USA), penicillin-streptomycin (Life Technologies; Grand Island, NY, USA), B27 (Life Technologies; Grand Island, NY, USA), hepes (Gibco Life Technologies; Grand Island, NY, USA), and L-Glutamine (Life Technologies; Grand Island, NY, USA). After a period of 6–8 days in culture, axons can be observed extending into the middle and distal chambers of the AIM device in sparse numbers to allow tracking of individual processes for subsequent experiments. Media composed of neurobasal media (Gibco Life Technologies; Grand Island, NY, USA), penicillin-streptomycin (Life Technologies; Grand Island, NY, USA), B27 (Life Technologies; Grand Island, NY, USA), and hepes (Gibco Life Technologies; Grand Island, NY, USA) is added every 3 to 4 days to maintain neuronal viability.

Cells in 2D cultures were trypsinized, centrifuged for 5 min at 180× g, washed with neurobasal media (GIBCO), and resuspended in tumorsphere media (50 mL neurobasal media + 1 mL B27 supplement + 500 µL Glutamax + 100 µL of penicillin/streptomycin + 100 µg FGF + 100 µg EGF), and 10⁵ cells were seeded in ultra-low attachment surface plates. Floating cells were transferred 24 h later into new ultra-low attachment surface plates for tumorspheres growth.
When they reach an appropriate size (five cells minimum), tumorspheres were concentrated by gravity in a 15 mL tube for 15 min. Then, tumorspheres were washed in PBS with trypan blue, centrifuged for 2 min at 5× g, resuspended in 500 µL PBS with a cut pipette tip, and moved into a 4-well plate. Images were analyzed with ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA.

For primary mouse neuronal cortical cultures, pregnant wild-type C57BL/6 mice were euthanized and decapitated after 17.5 days of gestation, and neurons were plated from dissected embryos of mixed sex as described previously (161 (link)). Briefly, brains were removed from isolated embryos, and dissected cortices were then moved into dissociation buffer (10 mM MgCl₂, 10 mM HEPES,1 mM kynurenic acid in Hank’s Balanced Salt Solution) supplemented with 16.67 U/ml papain (Worthington; LS003127), which was incubated for 45 min at 37 °C. Proteolyzed tissue was then rinsed in 10 mg/ml trypsin inhibitor (Millipore; T9253) twice for 5 min and then resuspended and mechanically dissociated in Neurobasal media (Thermo Fisher; 21103049) into a single cell suspension. Cultured cortical neurons were counted by hemocytometer, and 1.5 million cells were plated on 6-well plates pre-coated with 1 mg/ml poly-L-lysine dissolved in 1 M Tris pH 8.0. Cultures were maintained in Neurobasal media supplemented with 2% B-27 (ThermoFisher; 17504044), penicillin/streptomycin (100 U/ml and 100 μg/ml, respectively) (Millipore; 15140122), and 2 mM glutamine at 37 °C/5% CO₂. Culture media were changed to Neurobasal media without serum 3 days after plating and supplemented with fresh media every 4 to 5 days. All experiments were performed at days in vitro (DIV) 12.