Identification of Monocyte Subpopulations

In brief, cryopreserved peripheral blood mononuclear cell (PBMC) were thawed and surface-stained with the following antibodies to identify monocyte sub populations as previously described [17 (link)]: V500-conjugated anti-CD3, Qdot605-conjugated anti-CD14, Alexa700-conjugated anti-CD16, PE-Cy7-conjugated anti-CD56, PE-Cy7-conjugated anti-CD19, PE-Cy7-conjugated anti-CD20, APC-H7-conjugated HLA-DR monoclonal antibodies (mAbs), All antibodies were from BD Biosciences, except for Q605-conjugated anti-CD14 and yellow Live/Dead (Life Technologies). Stained PBMCs were acquired by flow cytometry, using a 4-laser custom BD-Fortessa instrument (Becton Dickinson) and analyzed with FlowJo software (Treestar Inc Ashland, OR) as previously described [17 (link)] (Fig 1).
The total monocyte count was calculated using white blood cell count (WBC) and percent monocyte values on the CBC conducted as part of entry evaluations on the same blood specimen as that utilized for flow cytometry in line with our previous reports [17 (link)].