Gating strategy to identify leukocytes and macrophage purity.

(A, B) Exemplary dot plots illustrating the gating strategy employed to identify the indicted (A) mouse or (B) human lymphoid cells. (C) Purity of the differentiated macrophages: human PBMC-derived monocytes were incubated for 7 days in RPMI1640 medium containing 10 ng/ml M-CSF for macrophage differentiation and the percentage of CD68+ macrophages was determined by intracellular staining. Left: representative flow cytometry data (blue = undifferentiated; red = differentiated); Right: Summary data (n = 3; un-diff = undifferentiated; diff = differentiated). (D-F) Evaluation of macrophage polarization. Primary human monocyte-derived macrophages were left unstimulated (UT) or stimulated for 12 h (D) with 100 ng/mL LPS and 20 ng/mL IFN-γ for M1 polarization (M1), (E) with 20 ng/mL IL-4 for M2a polarization (M2a), or (F) with 20 ng/ mL IL-10 for M2c polarization (M2c). Expression of indicated surface markers was analyzed by flow cytometry. The bar graphs indicate mean fluorescent intensity (MFI). The biological replicates of 6 independent donors pooled from 2 independent experiments are shown. (PDF)