Inubtf wskm b13i

hBMECs were maintained in human endothelial cell medium (no phenol red) supplemented with 0.1% VEGF, 0.1% heparin, 0.1% EGF, 0.1% hydrocortisone, 0.1% FGF, 1% l-glutamine, 1% antibiotic-antimycotic solution and 5% fetal bovine serum. Cells were cultured at 37 °C in humidified air containing 5% CO₂. Pre-cultured cells were plated at 1000 or 5000 cells in a glass-bottomed 96-well plate (5866–096, IWAKI) precoated with 0.3 mg/cm² gelatin (01393-100 ML, Sigma-Aldrich). After incubation for 24 h, medium was removed from each well and wells were refilled with new medium including DMSO (control) or Aβ₄₂ in DMSO and QDAβ. Time-lapse images were captured with an inverted microscope (Ti-E; Nikon equipped with a color CMOS camera (DS-Ri2; Nikon) and an objective lens (PlanApo λ 20 × /0.75 NA; Nikon) and standard TRITC (TRITC-A-Basic-NTE, ex: 532–552 nm, em: 594–646 nm) filter sets (Semrock). During observation, the cells were warmed in a chamber set at 37 °C (INUBTF-WSKM-B13I; Tokai Hit). Images were captured every 10 min and analyzed using NIS-Elements AR software (Nikon). The resulting data was edited by ImageJ software (NIH).

Z-stacks images were captured using an inverted microscope (Ti-E) and a confocal laser microscope system (C2 Plus; Nikon) equipped with an objective lens (PlanApo λ 20×/0.75 NA; Nikon, Plan Apo λ 100×/1.45 NA Oil; Nikon). Cells were maintained in RPMI supplemented with 5% FBS and 10% HS and warmed in a chamber set at 37 °C (INUBTF-WSKM-B13I; Tokai Hit) during observation. Images were captured and analyzed using NIS-Elements C software. For 3D time-lapse imaging, cells were maintained in DMEM/F12 (1:1) supplemented with 10% FBS and 4.5 ng/ml NGF, and warmed in a chamber set at 37 °C during observation. EGFP and QD were excited by a laser at 488 and 561 nm, respectively. Z-stack images were captured every 15 min and were oversampled by taking 1000 nm z-steps between acquired images and were analyzed using NIS-Elements C software. For observation of Aβ₄₂ aggregation by the MSHTS system, 25 μM Aβ₄₂ in and 25 nM QDAβ, PBS containing 5% EtOH and 3% DMSO were incubated in a 1536-well plate (782096; Greiner) warmed in a chamber set at 37 °C. Z-stack images were captured every 3 min and were oversampled by taking 2500 nm z-steps between acquired images and were analyzed using NIS-Elements C software.