Quantification of Candida - Phagocytes Interaction lusitaniae than at 1M:1Y MOI at T 24 h. Again the highest part of phagocytosing macrophages and macrophage cell death rates were obtained with C. albicans. Note the higher uptake of C. glabrata cells, and the escape from macrophages of the three Candida species at T 24 h. (B) Interactions of J774 macrophages and stationary-phase living yeasts at 5M:1Y MOI. Note that the macrophage killing with C. albicans was delayed compared to 1M:1Y MOI. Note the equally high uptake of C. glabrata and C. lusitaniae and the lower uptake of C. albicans biomass, that was the only one species that finally escaped the macrophages at that 5M:1Y MOI. For the legend description, please refer to Figure 3. doi:10.1371/journal.pone.0032621.g005 the yeast cells than they were at MOIs of 1N:1Y and 1N:5Y. In conclusion, while the association of the neutrophils with the three Candida species was similar and varied proportionally with the MOIs, neutrophil cell death was both similar and moderate in the presence of C. glabrata and C. lusitaniae. Candida albicans induced a higher neutrophil cell death, which varied proportionally with the MOIs. Discussion This study aimed to develop an improved in vitro model to finely quantify the cellular interactions between Candida yeasts and phagocytes. The method we developed is the first one that allows a semi-automated multi-parameter analysis for the simultaneous monitoring of two interacting populations in a host-pathogen context. The method was validated with three different Candida species and two types of phagocytes, the J774 murine macrophage cell line and human neutrophils. Yeasts were labeled with CFW, a fluorochrome that binds cell wall of live and dead fungal cells. CFW shows a relative insensitivity of its fluorescence to pH, which is an advantage in a phagocytic assay as the phagolysosomal acidification can modify the fluorescence of an internalized yeast [22]. The CFW was added in the culture medium at the beginning of the infection Figure 6. C. albicans, C. glabrata and C. lusitaniae escaping from macrophages. Macrophages were observed with the microscope after 5 hours of infection with C. albicans and 24 hours of infection with C. glabrata and C. lusitaniae. Yeast cells were stained with CFW (middle panel), macrophages were stained with calcein (right panel). See also video-microscopy of the interaction between J774 macrophages and C. albicans at a MOI of 1M:1Y (Movie S1), C. glabrata at a MOI of 1M:5Y (Movie S2) and C. lusitaniae at a MOI of 1M:2Y (Movie S3) over a 24-hour incubation period. The scale bar represents 5 mm. doi:10.1371/journal.pone.0032621.g006 PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e32621