β-Amyloid (22-35)

Hippocampal cells are cultured in DMEM supplemented with 10% (v/v) precolostrum newborn calf serum in poly-D-lysine-coated 24-well culture plates at 37°C under a gas mixture of 95% air/5% CO₂. After 6 days of culture, non-neuronal cell division is halted by 24 h exposure to 10^-5 cytosine arabinoside. After washing cell layers with DMEM, cells are cultured with 1 mL of DMEM per well in the absence of serum for 5 days before experiment. Cells are treated for 3 days with β-Amyloid (22-35) or its amidated (C-terminus) derivative by addition of 20 μL of stock solution (2 mg/mL of either in betaine buffer, pH 8.5, consisting of 0.7% betaine and 20 mM NH₄HCO₃) to 1.0 mL medium, unless otherwise specified. Control cells are treated with the betaine buffer alone. Morphological change of cells is checked throughout the course of experiment with a phase-contrast microscope. Dead cells are checked by trypan blue staining. Cell injury is assessed by measuring lactate dehydrogenase (LDH) activity released into medium during 3 days of treatment of neurons with peptides^[1]
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