Hcmec d3

Human cerebral microvascular endothelial cells (hCMEC/d3) (Fisher, Hampton, NH, USA) passage 30–40 were maintained in complete endothelium growth medium 2 (EGM2) (Lonza, Visp Switzerland) with 2% foetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) in a humidified incubator at 37 °C at 5% carbon dioxide (CO₂). Murine RAW 264.7 macrophages (ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified eagle medium (DMEM) (Invitrogen) containing 10% heat-inactivated FBS (Invitrogen) in a humidified incubator at 37 °C at 5% CO₂.

hCMEC/D3 (Fisher; passage 27–35), and HUVEC (passage 4–7, isolated as described [17 (link)]) cells were cultured using complete EGM-2 with 2% FBS, ECs were cultured in a humidified incubator at 37 °C at 5% carbon dioxide. For mechanistic experiments, ECs were treated with SR-BI blocking antibody (NB440–113 Novus, 1:500), the SR-BI inhibitor block lipid transport-1 (BLT-1, SigmaAldrich, 10 μM), the eNOS inhibitor L–NG-nitroarginne methyl ester (L-NAME, SigmaAldrich, 1 mM), the receptor associated protein (RAP, Oxford biome, 1 μM), the RAGE blocking antibody (176,902, R&D Systems, 1:50), heparin (10 mU), heparinase (SigmaAldrich, 0.2 mM) or the CD36 blocking antibody (JC63.1, ABCAM, 1:500) for 1 h before HDL priming. For miRNA experiments, cells were transiently transfected using Lipofectamine 2000 (Life Technology) 2 h before Aβ stimulation with the miR-223 mimetic (Life Technology, 4,464,066 (MC12301), 100 nM) or miR-223 inhibitor (Life Technology, 4,464,084 (MH12301), 100 nM) in EBM-2 containing 0.2% BSA.

The immortalized human brain microvascular endothelial cell line (hCMEC/D3, Merck, NJ, USA) was cultured in growth basal medium-2 (EBM-2, Lonza, Basel, Switzerland) with supplemented growth factors as previously described [12 (link)]. hCMEC/D3 cells were cultured on tissue culture flasks coated with collagen-I (10 μg/mL, rat tail, Sigma-Aldrich). Once confluency was reached, hCMEC/D3 cells were dissociated from the flask using Accutase™ cell dissociation reagent (Thermo Fisher, Waltham, MA, USA) for subsequent maintenance and seeding into microchannels.
Microchannels coated with and without 1% Teflon AF 2400 were incubated with 1:10 diluted Matrigel (Sigma, Saint Louis, MO, USA) solution overnight. hCMEC/D3 cells were detached from the cell culture flask and resuspended in the culture medium at a concentration of 1 × 10⁶ cells/mL, and the cell suspension was injected into microfluidic channels. Cells were cultured inside an incubator (5% CO₂ and 37 °C) for 2 days before being subjected to immunostaining or to CBD treatment under flow. A syringe pump equipped with a glass syringe and PEEK tubing was connected to the cell channel and perfused with CBD solution (1 μg/mL) at 2 μL/min for 3 h. Upon the completion of the experiments, the eluents were collected, and 10 μL of the collected CBD solutions were derivatized and analyzed by means of LC-MS/MS.