Cell culture

HeLa cells (ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), penicillin (100 unit/mL) and streptomycin (100 μg/mL). hTERT RPE-1 cells (ATCC, CRT-400) were cultured in DMEM/F12 (Gibco) supplemented with 10% FBS, 1% nonessential amino acids (Gibco), 2 mm sodium pyruvate (Gibco), penicillin (100 unit/mL) and streptomycin (100 μg/mL). Human umbilical vein endothelial cells (HUVEC) (Lonza, C2517A) were cultured in EGMTM-2 Endothelial Cell Growth Medium-2 BulletKit (Lonza, CC-3162). All cells were purchased from commercial sources and periodically tested for mycoplasma with DNA methods. Primary neurons were isolated from P0 to P1 mouse hippocampus using papain digestion and plated in Neurobasal A media (Invitrogen) with 2% Gem21 supplement (Gemini) and 10% FBS on glass-bottom culture dishes (Matek) coated with poly-d-lysine (20 μg/mL, Corning) and laminin 111 (1 μg/mL, Corning). Media was changed on the neurons after 4 h and maintained in serum-free Neurobasal A/Gem21 media containing 1% pen/strep and 2 mm l-glutamine.