Cd162 alexa fluor 647

Flow cytometric analysis was performed on a Fortessa or FACSCalibur FACS machine (BD). Standard staining was performed. In brief, cells were washed three times with FACS buffer and blocked using Fc-block CD16/32 antibody (BioLegend) for 15 min, followed by incubation with fluorescently labeled antibody for 30 min. Analysis was performed using FlowJo software (version 9.2; Tree Star). PBMCs were isolated by Ficoll centrifugation. The antibodies used were: CD11b-PE antibody (BioLegend), CD162–Alexa Fluor 647 (BD), CD62L-FITC and CD18-PE (eBioscience), and CD54-APC, CD14-Pe/Cy7, CD49d-FITC, CD11a-PE, CD11b–eFluor 450 (all BioLegend). Sterile FACS to maintain CD11b knockdown in CD11b^kd BV2 cell lines was performed after staining of CR3^kd and control cells with CD11b-PE antibody, as described in the previous paragraph. Cells were sorted on a FACSAriaIII cell sorter (BD) for CD11b^low-expressing cells, defined by CD11b expression of <80% of control cells. Then, cells were cultured as described in the Cell lines section.