Flow cytometry gating strategy to identify the three monocyte subsets from PBMCs.

(A) FSC x SSC gating to obtain mononuclear cells based on size and granularity. (B) Live/dead Aqua marker used to identify live cells (Aqua negative cells). (C) Staining with lineage-specific antibodies against T-cells, B-cells, NK cells, Dendritic cells and neutrophils (‘dump’, see Methods) to allow exclusion of these cells. (D) HLA-DR was used to identify monocytes, which are HLA-DR positive. (E) CD14 x CD16 gating used to select monocytes. (F) Gates for monocyte subsets based on CD14 and CD16 expression were determined using respective Fluorescence minus one (FMO) controls.