C2519as

Human iPSC-ECs (Cellular Dynamics International, CDI) and HBMECs (Angio-proteomie, cAP-0002) were cultured on flasks coated with human fibronectin (Millipore) (30 µg mL⁻¹) in vascular medium (VascuLife VEGF Medium Complete Kit, Lifeline with iCell media supplement, CDI). Human primary astrocytes and pericytes (ScienCell) were cultured on flasks coated with poly-L-lysine in culture medium (ScienCell), and maintained in a humidified incubator (37 °C, 5% CO₂). HUVECs (Lonza, C2519AS) were cultured in EGM-2 media (Endothelial Cell Growth Medium-2 Bulletkit, Lonza). Culture medium was replaced every 2 days and cells were used between P3 to P5.

Primary HUVECs from pooled donors (Lonza, C2519AS) were cultured in tissue culture vessels coated with 0.2% gelatin type B (Sigma-Aldrich) containing Clonetics EGM-2 Complete Media (Lonza). Cells between passages three to five were used in all experiments. RAW264.7 murine macrophages (American Type Culture Collection, TIB-71) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). HEK-Blue cells, which stably express CD14, MD2, a NF-κB reporter, and TLR4 or TLR2, were obtained from InvivoGen. The cells were cultured in 96-well plates in complete DMEM containing 10% Endo-low FBS to 70% confluency and were treated with LPS or LTA for 16 hours. Cell culture medium (100 μl) was then mixed with 100 μl of HEK-Blue Detection Medium and incubated at 37°C for 1 hour. LPS and LTA activate TLR4 and TLR2, respectively, resulting in induction of NF-κB reporter expression, which catalyzes the conversion of the HEK-Blue Detection Medium to a blue color, which was quantified by measuring the absorption at 650 nm. All incubations were performed in a 37°C incubator under 5% CO₂ atmospheric conditions.