Anti ccr2 pe

Primary BMVECs were stimulated for 4–18 h with 10 ng/mL TNFα + IFNγ ± BETi or DMSO, followed by staining with FITC anti-VCAM1 and APC anti-E-selectin antibodies (BD™ Bioscience). THP-1 were treated with DMSO or BETi for 48 h followed by staining with anti-CCR1 Alexa Fluor^® 647, anti-CCR2 PE, anti-CCR5 FITC, anti-CXCR2 FITC, anti-ITGA4 BV421 or anti-IGTAM APC antibodies or isotype controls (BD™ Bioscience). Fluorescence was quantified using BD FACSCelesta™ Flow Cytometer. Mean fluorescence intensity (MFI) and % positive cell numbers were calculated with FlowJo™. The concentrations of MCP-1 and IL-6 in the tissue culture supernatant were measured using a BD™ Cytometric Bead Array Flex Set.

Peripheral blood mononuclear cells (PBMC) were obtained from K2EDTA blood samples. Tubes were centrifuged and plasma was collected, aliquoted and stored at −20 °C. Cells were diluted with phosphate-buffered saline (PBS), PBMC were isolated by Ficoll density gradient and directly used for flow cytometry (FC) analysis and monocyte isolation.
Monocyte phenotype was analyzed by FC (FACS Canto II, BD Bioscience, San Jose, CA, USA). PBMC were stained with anti-CD3-V450, anti-CD19-V450, anti-CD56-V450, anti-CD14-FITC (BD Bioscience, San Jose, CA, USA), anti-CD33-PE-Cy7 (eBioscience, San Diego, CA, USA), anti-HLA-DR-APC, anti-CD16-V500, anti-CCR2-PE, anti-CCR5-APC-Cy7 and anti-CD86-PerCP-Cy5.5 (BD Bioscience, San Jose, CA, USA). Classical monocytes were defined as CD14+CD16−, intermediate monocytes as CD14+CD16+ and non-classical monocytes as CD14−CD16+. FC data were analyzed in FlowJo V10.