Hla dr pacific blue l243

BAL cells were stained using fluorochrome-conjugated mouse monoclonal antibodies directed to human CD11b-PE (clone D12), CD45-PerCP (TU166), CD20–PE-Cy7 (L27), CD16-AF647 (3G3), CD3–APC-Cy7 (SP34-2),CD8-AmCyan (SK1), CD95-PE (DX-2), CD69-PerCP (FN50), CCR4–PE-Cy7 (1G1), CD28-APC (CD28.2), and CD4-V450 (L200) (All from BD Biosciences), as well as HLA-DR–Pacific Blue (L243; BioLegend). Fluorescence was measured on a FACS Canto II and analyzed using BD FACS Diva software (both BD Biosciences) and FlowJo software (Tree Star Inc.) (Supplemental Methods, Supplemental Figure 4). Extrapolated cell number was determined for each of the analyzed cell types present in BAL fluid as follows: “number of BAL cells/mL” × “frequency of the population.”

Immunofluorescent staining of DC surface antigens was performed using a panel of fluorescence-conjugated monoclonal antibodies and analyzed by flow cytometry: CD80 (BV510, 2D10), CD83 (PerCP-Cy5.5, HB15e), CD86 (APC, BU63), and HLA-DR (Pacific Blue, L243, all BioLegend). DCs could be separated from non-differentiated monocytes according to their higher FSC/SSC intensities. Mean fluorescence intensity (MFI) ratios were determined by dividing the MFI value measured for the antibody by that of the corresponding isotype control. In parallel, IL-6 secretion into the supernatant was quantified via cytometric bead array (BD Biosciences) according to the manufacturer’s instructions.