Crl 1721

In a Petri dish, PC-12 cells were cultured for one week prior to use. PC-12 cells were maintained in an incubator at 37 °C/5% CO₂, and cells were fed every other day using F12-K medium supplemented with 5% (v/v) fetal bovine serum (FBS), 10% (v/v) heat-inactivated horse serum (HS), and 1% (v/v) penicillin–streptomycin solution. All experiments were conducted on undifferentiated cells plated in 96-well plates at a density of approximately 5 × 10⁴ cells/mL for the ATP assay [24 (link)]. On the first day of use, cells were harvested by first adding 2 mL trypsin and then incubated for 15 min. Next, 2 mL of fresh medium was used as a wash, and the cells were centrifuged and re-suspended. Cell density was measured by cell counting using a hemocytometer and a tissue culture microscope. After cell density was determined, 96-well plates and 6-well plates were plated with 2.5 × 10⁴ cells per well and 5 × 10⁵ cells per well for the Adenosine 5′-triphosphate (ATP) assay and Western blot analysis, respectively. Cells were incubated for 24 h, and varying Carbamathione concentrations were added along with 10 mM glutamate/hypoxia at 24 h and reoxygenation at 24 h.
Cell lines:
The cell line was purchased from ATCC (cat # CRL-1721.1) and was last validated and certified in 2018.
The cell line is used up to a maximum number of five passages.

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.