Cell experiment:

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℃ 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].

β-Amyloid (22-35) is a 14-aa peptide, shows aggregates and induces neurotoxicity in the hippocampal cells.

β-Amyloid (22-35) shows significant cytotoxicity on the rat hippocampal neurons at 40 μg/mL, but exhibits no cytotoxic effect on the glial cells[1].

[1]. Takadera T, et al. Toxic effect of a beta-amyloid peptide (beta 22-35) on the hippocampal neuron and its prevention. Neurosci Lett. 1993 Oct 14;161(1):41-4.