Alzheimer's Disease Modeling in PC12 Cells

Pheochromocytoma (PC12) adherent cells (ATCC^® CRL-1721.1™) were proliferated in RPMI-1640 (ATCC), which was supplemented with 10% horse serum (HS), 5% fetal bovine serum (FBS), and 1% pen/strep on collagen (50 μg/mL) at 37 °C and 5% CO₂. Cells were differentiated using nerve growth factor (NGF, 200 ng/mL, Millipore, Burlington, MA, USA) in RPMI-1640, 2% HS, 1%FBS, and 0.2% pen/strep for 24 h before treating with 100 nM Aβ₄₂. Soluble oligomeric Aβ₄₂ peptides were prepared as described in Arora et al. [15 (link)]. These preparations were shown to yield a stable oligomeric form of Aβ₄₂. Daily media and treatment changes ensured consistent exposure to Aβ₄₂ [16 (link)]. After three days, cells were solubilized by the addition of a 0.1% Triton X-100 lysis buffer (Triton X-100, 1 M Tris HCl, 1.5 M NaCl, 0.25 M EDTA, and 10% glycerol, in the presence of protease inhibitors (Complete Mini, Roche, Basel, Switzerland) in 10 mL of lysis buffer) as described [17 (link)]. The protein concentration was determined by a Bradford assay.