Anti human cd54 apc

Normal Human Bronchial Epithelial Cells (NHBE) and Bronchial Epithelial Cell Growth Medium were obtained from Lonza (Switzerland). Primary Human Nasal Epithelial Cells (HNEpC) and Airway Epithelial Cell Growth Medium were obtained from Promocell (Germany). Grindelic acid (purity ≥95% (LC/MS-ELSD)), clarithromycin (purity ≥95% (HPLC)), budesonide (purity ≥98% (HPLC)), LPS (from Escherichia coli 0,111:B4), Ficoll Hypaque gradient, GM-CSF, HEPES buffer, l-glutamine, RPMI 1640 medium, Fetal bovine serum (FBS), Cell Dissociation Solution (non-enzymatic), Accutase™, propidium iodide and Calcein-AM were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). TLR-4-(PE)-conjugate (cat no. HTA125) was obtained from eBioscience. Human Quantikine ELISA Kits were purchased from R&D System (Minneapolis, USA). Anti-Human CD54-(-APC) (cat no. 559771), Anti-Human CD106-(-PE) (cat no. 555647), Anti-Human CD62E-(-PE) (cat no. 551145) were purchased from BD Pharmingen.

For cell surface and intracellular staining, standard protocols were followed using monoclonal antibodies. Cells and antibodies were co-incubated on ice in the dark for 30 minutes. For intracellular staining of cytokines, cells were permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA) for 30 minutes on ice, followed by an additional 30-minute incubation with antibodies in the dark at 4°C. The following antibodies were used: Anti-HLA-DR-APC (BD, cat. no.: 339194), anti-human CD54-APC (BD, cat. no.: 561899), anti-human CD86-FITC (BD, cat. no.: 560958), anti-human CD3-Percp (BD, cat. no.: 552851), anti-Human CD4-PE (BD, cat. no.: 557344), anti-Human CD8-FITC (BD, cat. no.: 555366), anti-human Ki-67-APC (BioLegend, cat. no.: 350514), and anti-IFN-gamma-APC (BD, cat. no.: IC285A-100). Imaging flow cytometry was performed using the ImageStreamX MarkII quantitative imaging flow cytometer, while conventional flow cytometry was performed using the BD FACS Calibur flow cytometer.