Macrophage Immunophenotyping by Flow Cytometry

At 24 h post transfection, macrophages were harvested by scraping, then washed with cold autoMACS running buffer (Miltenyi Biotec). Cells were incubated with FcR blocking reagent (Miltenyi Biotec) for 10 min at 4°C, to avoid unspecific antibody binding. Upon washing, cells were stained with CD80-PE (clone L307.4) (BD Bioscience, Heidelberg, Germany) antibody with dilution factor 1:100 (5 μg/mL final concentration) at 4°C for 10 min. After a final washing step, cells were analyzed with MACSQuant VYB (Miltenyi Biotec). For live-dead discrimination, DAPI, at a final concentration of 1 μg/mL, was added to each sample immediately before measurement. All flow cytometric data were analyzed by FlowJo software V10 using the previously established gating strategy.⁴¹ (link) Briefly, cells were initially identified from debris by gating on forward versus side scatter area (FSC-A versus SSC-A) dot plots, followed by exclusion of aggregated cells using forward scatter area against height (FSC-A versus FSC-H). DAPI-negative cells were identified as live cells. EGFP-positive cell populations were determined among live single cells, by gating with respect to untransfected negative controls.