Rencell cx human neural progenitor cells

ReNcell CX human neural progenitor cells (Millipore, Billerica, MA) (NPCs) were cultured according to the manufacturers' directions. Cells were seeded on laminin‐coated glass coverslips (20 μg/ml) at a density of 5000 cells per cm² in Rencell media, or for lysine restriction experiments in custom lysine‐free DMEM‐F12 (3:1) supplemented with B27 (2%), 2 μg/mL Heparin, FGF (20 ng/ml), and EGF (20 ng/ml). For experiments analyzing effects of high levels of lysine, media was supplemented to final lysine concentration 1.18 mg/mL (10 times the usual concentration in 3,1 DMEM‐F12 media). For experiments analyzing effects of high PN, PN was supplemented to 18 mg/mL PN (9 times the usual cell culture media concentration).
Cells were harvested for metabolomics analysis with modifications of published protocols.²⁰ In brief, coverslips were removed from cell culture wells using forceps, and washed briefly in 37°C ddH₂0, then placed in ice cold 50% MeOH in a 6‐well‐plate kept on ice. Cells were scraped into the 50% MeOH and transferred to 1.5 ml tubes and stored at −20°C until further processing. Samples were then sonicated for 15 seconds (Bandelin Sonorex) and centrifuged for 15 min, 4°C at 14500 rpm following DNA quantification by Hoechst assay.