HT22 Cell Culture and Treatment

HT22 cells, a mouse hippocampal cell line obtained from the immortalization of primary hippocampal neurons using a temperature sensitive SV40 T-antigen and subcloned from HT4 cells based on sensitivity to glutamate (Davis and Maher, 1994), were obtained from Dr. Dave Schubert (Salk Institute, La Jolla). The cells were grown in 75 cm² culture flasks in high-glucose Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% heat inactivated (HI) fetal bovine serum (FBS), 12 mM NaHCO₃, 5 mM HEPES and 100 μg/mL penicillin-streptomycin, pH 7.3 in a humidified incubator with 5% CO₂ and 95% air at 37°C. When 80% confluency was reached, cells were detached using a Ca²⁺-Mg²⁺-free dissociation medium containing 140 mM NaCl, 8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 1.47 mM KCl, 0.55 mM EDTA, pH 7.3 and then centrifuged at 800 rpm for 5 min and subsequently sub-cultured or plated at a density of 0.005 × 10⁶ cell/well in 96-multiwell plates. Twenty four hour after plating, cells were treated with 1 μM AβO in the presence or absence of 1, 10 or 100 μM boldine in culture conditioned medium (medium where the cells were plated) for 24 h at 37°C in a humidified culture chamber containing 95% O₂ and 5% CO₂. The effect of boldine alone was also tested.