Cd106 pe

To analyze trafficking of dye-labeled lymphocytes, tissues were digested and stained with the following antibodies: CD45 BUV395 (BD), CD45 Pacblue (Biolegend), CD11c FITC (Biolegend), CD4 BV711 (Biolegend), CD8 PE (eBioscience), CD8 PE-Cy7 (eBioscience), and CD19 BV510 (Biolegend). For intracellular chemokine staining of CXCL16 (R&D Systems) was performed using the FoxP3 intracellular staining kit (eBioscience); mice were treated i.v. with Berfeldin A (Sigma) for 4 h prior to harvest. For vascular adhesion molecule and chemokine expression a combination of the previous antibodies were used as well as CD31 PE (eBioscience), CD54 FITC (eBioscience), and CD106 PE (eBioscience). All antibody staining was done for 30 min on ice. Samples were collected on either a BD LSRII or LSR Fortessa and analyzed by FlowJo.

Cells were digested with trypsin and washed with cold PBS, and then stained with isotype IgG or the following monoclonal antibodies: CD106-PE, CD36-PE, CD90-FITC, CD105-APC, CD73-FITC, CD34-PerCP, CD45-FITC, CD127-FITC, CD31-FITC (all from eBioscience). After being washed by PBS, the labeled cells were resuspended in PBS supplemented with 0.2% FBS and at least 105 events were acquired using BD Accuri™ C6 Flow cytometer (BD, NJ, USA).

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.