Cd54 apc

For flow cytometry, the following fluorochrome-conjugated monoclonal antibodies were used. BD Biosciences: HLA-DR-APC (Clone: G46-6), CD86-FITC (Clone: FUN-1), CD80-PE (Clone: L307.4), CD54-APC (Clone: HA58), CD25-FITC (Clone: M-A251), CD127-BV421 (clone HIL-7R-M21), IFN-γ-FITC (Clone: 4S.B3), IL-4-PE (Clone: MP4-25D2); eBioscience: FoxP3-APC (Clone: 236A/E7), IL-17A-PE (Clone: Ebio64cap17); Beckman Coulter: CD40-PE (Clone: MAB89); Biolegend: CD4-PerCP (Clone: SK3). Cell viability was detected using the fixable viability dye eFluor 506 (eBioscience).
Antigen affinity-purified polyclonal anti-human TLR4 goat IgG was purchased from R&D systems. Cytokines (recombinant human granulocyte-macrophage colony-stimulating factor and IL-4), MicroBeads (CD14 and CD4) and cell purification units were obtained from Miltenyi Biotec. Protein-A agarose beads were from Cell Signalling Technology, and E. coli 055:B5 LPS and Polymyxin B-conjugated agarose beads were from Sigma-Aldrich. TLR4 signaling inhibitor CLI-095 and CpG ODN 2006 were procured from InvivoGen.

hCMEC/D3 cells (21 (link)) were cultured and stained as described previously (Williams et al., 2018). Briefly, cells were stimulated for 24 hours with human TNF (100 ng/ml, R&D Systems, Minneapolis, MN, USA) in presence or absence of Atrosimab (100 µg/ml). Then cells were harvested and collected in PBS containing 0.02 mM EDTA and 0.4 mg/ml collagenase (Sigma-Aldrich). Cells were then stained with antibodies against either human VCAM-1 (CD106-PE, STA, eBioscience) or ICAM-1 (CD54-APC, HA58, eBioscience). Cells were then fixed in 1% PFA and kept in the dark until analysis. Flow cytometry acquisition was performed on a FACSCanto II (BD Biosciences) using the BD FACSDiva software. All data was analyzed using FlowJo software.