Investigating Amyloid Toxicity in Neuroblastoma

SH-SY5Y cells, a human neuroblastoma cell line (ATCC, Manassas, VA, USA), were cultured in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Sigma-Aldrich) and 100 μg/mL of penicillin-streptomycin (Gibco) at 37°C in 5% CO_2. iPS-MLs were maintained as described previously [30 (link)]. Briefly, iPS-MLs were cultured in MEMα medium (Wako) supplemented with 10% FBS, 100 μg/mL of penicillin-streptomycin, 25 ng/mL of human M-CSF (Prospec-Tany Technogene, Rehovot, Israel), and 50 ng/mL of human granulocyte M-CSF (Prospec-Tany Technogene) at 37°C in 5% CO_2. To inhibit proliferation of cultured iPS-MLs, mytomycin C (Sigma-Aldrich) pre-treated iPS-MLs (1 × 10⁴, 5 × 10⁴, or 1× 10⁵ cells/well) or SH-SY5Y cells were cultured with 800 nM native (wild-type or mutated) or aggregated TTR in 96-well flat-bottomed plates in FBS-free conditions. Three days later, culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) and western blotting, and cultured cells used for immunohistochemistry.