Fitc aβ42

SMC and EC were seeded in 6-well plates at a concentration of 300,000 and 500,000 cells per well, respectively. After two days in culture, cells were incubated with 0.1 μM of FITC-Aβ42 (Bachem) in the absence or presence of 200 μg/mL of HDL or BSA in respective growth media. After 3 or 24 h, cells were washed twice with PBS, detached using 0.25% trypsin for 5 min at 37 °C before adding RPMI to collect the cells. Floating cells were pelleted by centrifugation (300 g) for 5 min, washed four times with FACS buffer (PBS containing 2% FBS, 1 mM EDTA and 0.1% sodium azide), and suspended in a final volume of 200 μL of FACS buffer and counted immediately using a BD™ LSRII flow cytometer. Data were analysed using FlowJo software (RRID:SCR_008520).

ECs were seeded at 3 × 10⁵ cells/well in 12-well plates and cultured until confluent for 2 to 3 days. On the day of the assay, ECs were primed for 2 h with 100 μg/mL HDL before stimulating with 0.1 μM of Aβ40 and Aβ42 monomers at 37 °C for total association, or at 4 °C for cell surface binding. After 3 h, hCMEC/D3 were washed 3 times with PBC and lysed in RIPA buffer containing 10 mM Tris pH 7.4, 150 mM NaCl, 1.0% NP-40, 1.0````% sodium deoxycholate, 0.1% SDS and cOmplete protease inhibitor with EDTA (Roche). Aβ40 (KHB3442, Life Tech) and Aβ42 (KHB3482, Life Tech) were quantified using commercial ELISAs and normalized to total protein concentration. For Aβ uptake, hCMEC/D3 were seeded at 1 × 10⁵ cells/well in 24-well plates and cultured to confluence for 2 to 3 days. On the day of the assay endothelial, ECs were primed for 2 h with 1 mg/mL of HDL before stimulating with 1 μM monomeric FITC-Aβ40 and FITC-Aβ42 (Bachem) prepared as described above. After 3 h at 37 °C, hCMEC/D3 were washed 3 times with PBS and fixed in 4% PFA for 20 min. After one Tris-HCl and two PBS washes, hCMEC/D3 were mounted in Prolong antifade reagent.