Quantification of Candida - Phagocytes Interaction Figure 2. Flow cytometry analysis of the macrophages interacting with yeasts. C. lusitaniae cells and macrophages were stained as described in the Materials and Methods. Histograms plot the cell size (SSC) against cellular complexity (FSC) or the number of cells against the fluorescence intensity. The vertical lines define the baseline above which the fluorescence is positive, and was gated using each labeled population alone, and unstained cells as negative controls. Thus, the CFW-labeled yeast cells alone only showed a positive signal for CFW fluorescence (A), the calcein and anti-CD16 double-stained macrophages alone only showed a positive signal for calcein and anti-CD16 fluorescences (B). The anti-CD16 fluorescence was plotted against the calcein fluorescence to gate the population Q2, corresponding to alive double-stained calcein+anti- CD16+macrophages. When the calcein and anti-CD16 double-stained macrophages were infected with CFW-labeled yeasts (C), the analysis of the population Q2 for CFW fluorescence distribution after 5 hours of incubation showed two distinct cells subsets: Q2-2 corresponding to phagocytosing macrophages, and Q2-4 corresponding to non-phagocytosing macrophages. The scale bars on the microscopy panels represent 5 mm (A and upper panel in C) or 30 mm (B and lower panel in C). doi:10.1371/journal.pone.0032621.g002 PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e32621