Transwells 24 well pore size

The U251 cells were inoculated under the chambers of Transwells (24-well; pore size: 3 μm; Corning, USA) (1 × 10⁶ cells per well). After U251 cells adherence, the bEnd.3 cells were inoculated into inserts (1 × 10⁵ cells per well), so that the endothelial cell line could contact with the glioblastoma astrocytoma cells through the micropores³⁷ (link). The culture medium was replaced every 1–2 days. The integrity of this bilayer was monitored using transendothelial electrical resistance (TEER) measurements (Millicell-ERS, Millipore, USA). After the transmembrane resistance of the model reached 200 Ω, added new medium containing Aβ (5 μmol/L) into corresponding wells. After 24 h, replaced the medium and added different nanoparticles labeled with coumarin-6 into the chambers of Transwells. 4 h later, the confocal microscope was used to observe the imaging of this bilayer.

For invasion assay, 4T1 cells were firstly starved in RPMI 1640 medium (without serum) for 12 h before seeded into the upper chambers of transwells (24-well; pore size: 8 μm; Corning, USA) with a density of 3 × 10⁵ (Transwells were precoated with Matrigel, 25 μg/well, BD Bioscience). Then, another 100 μL of nanoparticles (final concentration: AuNCs, 2 μg/mL; H₂O₂, 2 μmol/L) in serum-free medium were added to each upper chamber, while the lower chambers were filled with 600 μL RPMI 1640 medium containing 20% FBS as chemoattractant. After incubation in hypoxic environment for 24 h, cells were fixed with 4% paraformaldehyde and stained with crystal violet for 20 min. Cells in the upper chambers were removed by a cotton swab, and the invaded cells to the lower side of membrane were imaged by an inverted microscope. For further quantitative measurement, the crystal violet was dissolved in 33% acetic acid, and the absorbance was detected at 570 nm by microplate reader (Thermo Scientific, Varioskan Flash, USA).