active metabolism and membrane integrity, and anti-CD16 antibodies which stained the membrane. To determine the rate of yeasts internalized in phagocytes, we exploited the ability of the trypan blue, incapable of penetrating into viable phagocytes [5], to quench the fluorescence of the extra-phagocyte CFW-labeled yeasts, in order to detect solely the CFW fluorescence of the internalized yeasts [6]. Microscopy is commonly used to count the number of phagocytes associated to yeasts as well as the number of yeasts internalized within phagocytes, or cell survival using vital dye exclusion [7–8]. However, microscopy may be too cumbersome for analyzing a large number of strains. Several studies described the use of flow cytometry or fluorometry to measure the association of the phagocytes to the yeasts, and the uptake of yeasts by phagocytes [9,10,11,12,13,14,15]. Therefore, we chose to use flow cytometry and modified a previously published assay [6] for the kinetics studies of the proportion of phagocytes associated to yeasts (either simply attached to the membrane or internalized) and phagocyte survival when infected by yeasts (compared to uninfected phagocytes). In this work, we describe an improved in vitro model of phagocyte infection with yeast, that allows an accurate quantifi- cation of both cell types (phagocytes and fungal cells) interaction and outcome during a single infection experiment up to 24 hours, and suitable for high-throughput screening of small phenotypic differences. As a proof of principle, we compared to which extent three species of Candida, exhibiting different morphological types, could differ in their interaction with murine macrophages and human neutrophils. We chose Candida albicans, which can develop under the form of yeast, pseudo-hyphae and true hyphae, Candida lusitaniae, found as yeast and pseudo-hyphae forms, and Candida glabrata only existing as a unicellular yeast form. We demonstrated that our model was sensitive enough to point out differences of interaction between those three Candida species and two types of phagocytic cells, and practical to analyze a very large number of each cell type over a 24-hour interaction. Results Fluorometry and Flow Cytometry Allow an Accurate Quantification of the in Vitro Candida - Macrophage Interaction The challenge in developing an in vitro cellular model to perform kinetics studies relied on the fact that the two interacting populations changed over time: the fungal cells grew and divided, leading to an overall increase in the fungal biomass, and the infected phagocytes eventually died, imposing the need to specifically mark each population to monitor their outcome during the interaction. Accordingly, the fungal cells were continuously labeled with Calcofluor White (CFW) which binds chitin and glucans in the cell wall of both live and dead yeast [16]. Because of the morphological changes that could occur for the different yeast species during the infection process, variations in the CFW fluorescence were interpreted as variations in the fungal biomass rather than variations in the number of cells. The murine macrophages of the J774 cell line were double stained with calcein, a marker of active metabolism and membrane integrity, and anti- CD16 antibodies which stained the membrane; the phagocytes that were fluorescent for both markers were considered to be alive. We checked that CFW, the only stain present during the infection process, did not alter the growth or viability of yeasts and phagocytes at the concentrations used (Figures S1 and S2). Furthermore, using the microscope and trypan blue as a vital dye, we checked that CFW was neutral for macrophages viability, and Quantification of Candida - Phagocytes Interaction that alive macrophages, intensely fluorescent for calcein, did not accumulate trypan blue (data not shown). Further controls were performed to determine and validate the experimental conditions that were used to analyze the interaction between the two stained cell types by fluorometry. First, we verified that calcein fluorescence was proportional to the number of macrophages (Figure S3). Moreover, in order to quantify the multiplication of the fungal biomass, total fluorescence of CFW- labeled yeasts was measured in PBS. We established that CFW fluorescence was linear with the OD600 up to 24 hours for the three Candida species when inocula up to 16106 CFU per 200 ml in the microplate well were used to infect macrophages. Thus, CFW fluorescence intensity is proportional to the fungal biomass (Figure S4). Accordingly, all infection experiments were performed using yeast inocula of 66104 CFU or 16105 CFU depending on the MOI (Multiplicity Of Infection). The trypan blue quenching assay, based on the extinction of the fluorescence of extracellular yeasts (i.e, free yeasts and yeasts adhering to phagocytes but not internalized), was used to determine the percentage of the total fungal biomass that was phagocytosed by macrophages. First, we checked that trypan blue efficiently quenched the fluorescence of free CFW-labeled yeasts (Figure 1A): C. lusitaniae cells were labeled with 5 mg/ml CFW. CFW emits a fluorescence around 430– 460 nm when bound to the yeast cell wall and gives negligible background fluorescence when unbound. When 250 mg/ml trypan blue was added to the CFW-labeled yeasts, the CFW fluorescence was quenched to the baseline observed for CFW alone. Using the microscope, we confirmed the quenching of the membrane-bound CFW-labeled yeasts (Figure 1B). Then, we determined the concentrations of trypan blue that were necessary to quench the CFW fluorescence emitted by up to 16107 yeast cells of the three species used in this study : 250 mg/ml of trypan blue was sufficient to quench the CFW fluorescence of both C. lusitaniae and C. glabrata, whereas 1 mg/ml was needed for C. albicans (e.g. Figure 1C for C. lusitaniae). We used flow cytometry in order to accurately quantify the association of macrophages with the fungal cells (in other words the percentage of phagocytosing macrophages) and assess the viability of the phagocytes. The CFW-labeled yeasts and the anti-CD16 and calcein double-stained macrophages were observed as distinct populations on the flow cytometer (Figure 2A and 2B). When infected with yeasts (Figure 2C), two distinct subsets of macrophages could be distinguished among the alive macrophages (quadrant Q2): the macrophages associated with yeasts (membrane-bound and/or ingested), i.e phagocytosing macrophages, positive for CFW fluores- cence (quadrant Q2-2), and non-phagocytosing macrophages, negative for CFW fluorescence (quadrant Q2-4). The different populations were sorted at T 5h and observed with the microscope to verify that the cells subset that was positive for calcein, anti-CD16 and CFW fluorescence truly corresponded to the macrophages that had taken up the yeast cells (Figure 2C). The percentage of macrophage viability was calculated as the number of macrophages positive for both fluorescence (calcein and anti-CD16) in an infection assay versus the uninfected macrophages control. The term ‘‘aggressiveness’’ was used to qualify the effect of the yeast cells on macrophage viability. Macrophages are Less Efficient to Control Candida Albicans than C. Lusitaniae or C. Glabrata Infection in Both the Stationary and Exponential Phase at a MOI of 1M:1Y It is known that the yeast cell wall composition and transcript profile vary with the physiological state of development [17], notably when considering cells grown in the stationary vs. PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32621