Anti cd14 pacific orange

The levels of membrane markers were characterized by flow cytometry using fluorescently labelled antibodies. On day 0 and 5, nonadherent cells were harvested and collected by centrifugation. Cells were resuspended in 100 µL of culture media with antibodies, and the cells were incubated at room temperature for 20 min in the dark. Afterward, cells were diluted with 400 µL of PBS and analyzed using an Attune NxT flow cytometer. The following monoclonal antibodies were used: anti-CD14 Pacific Orange (ThermoFisher Scientific, MA, USA), anti-DC-SIGN APC/Fire 750, anti-CD80 AF 488, anti-CD83 APC/Fire 750, anti-CD86 Pacific Blue, anti-HLA-DR PerCP, anti-CD85k APC, anti-CD207a PE (All from Biolegend, San Diego, CA). For the purpose of isotype control, an IgG1, IgG2a and IgG2b cocktail was used (all from Biolegend, San Diego, CA). The concentration of cells in a suspension was 1,000,000 per millilitre. Firstly, 150 µL of suspension of dendritic cells were transferred into a new one-well culture slide. The next step was washing cells with sterile PBS twice and then adding the solution of 1. This volume was 5 µL (for concentration 10 -5 M) and 0.5 µL (for concentration 10 -7 M). Cells were ready for analysis with fluorescent microscope after 15 or 30 min of incubation.