Cell labeling was performed in a round-bottomed 96-well microtiter plate using 5 × 105 leukocytes per well as previously described [38 (link)]. All incubation and centrifugation steps were performed at 4 °C. Separated leukocytes were incubated with unlabeled primary monoclonal antibodies (mAbs) (Supplementary Table S1 ) specific for the cell surface molecules CD4, WC-1, CD14, CD163, CD172a, MHC-II, CD11a, CD18, CD44, and CD45R [15 (link),34 ,35 (link),36 (link),37 (link)] for 15 min in the dark. After two washings in staining buffer, the cells were incubated with fluorochrome-labeled anti-mouse IgM, IgG1, and IgG2a secondary antibodies (Invitrogen, Schwerte, Germany) for 15 min in the dark. Parallel setups were incubated only with antibody isotype controls. After two washings, labeled cells were analyzed on an Accurie C6 flow cytometer (BD Biosciences, Heidelberg, Germany) by the acquisition of at least 100,000 total leukocytes. Collected flow cytometric data were analyzed using the CFlow Software (V 1.0.264.21; BD Biosciences, Heidelberg, Germany). Leukocyte count was estimated under a microscope using the Neubauer counting chamber after staining of the blood sample with Türk Solution.
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