labeled yeasts. After washing the wells with PBS, 200 ml of PBS was added and the CFW fluorescence was measured. For each well, the value of the fluorescence after quenching was compared to the total fluorescence in PBS in order to determine the relative amount of yeasts internalized over time. Uninfected macrophages were treated in the same manner as the infected ones to assess the residual fluorescence not attributable to the yeasts before and after trypan blue quenching. Yeasts alone were included in the plate and treated in the same way in order to validate the efficiency of trypan blue quenching. To follow the multiplication of the fungal biomass in the presence of macrophages along the infection, we compared the total CFW fluorescence values at T 5 h and T 24 h to the initial value at T 30 min and determined the biomass multiplication factors. Flow Cytometry Analysis The quantification of the attachment and/or ingestion of the yeasts by the phagocytic cells was done by flow cytometry analysis using a FACSCanto II (Becton Dickinson) equipped for Calcofluor White (lex 365 nm, lem 430 nm,), calcein-AM (lex 496 nm, lem 516 nm) and anti-CD16-APC (lex 600 nm, lem 630 nm) fluores- cence measurements. The phagocytic cells were infected by CFW- labeled yeasts as described above. After different periods of incubation, the plate was kept on ice to stop phagocytosis. Supernatants containing free yeasts were eliminated, and after a PBS wash and trypsin treatment, the phagocytic cells were labeled with 0.2 mg/ml anti-mouse CD16-APC (Beckman Coul- ter) and 0.2 mM calcein-AM (Sigma) and the samples in the 96- well plates were analyzed by flow cytometry. A constant volume (60 ml) of each sample was measured at a high flow rate (2 ml/s). The data were collected using a linear representation for the side scatter (SSC) and forward scatter (FSC) and a logarithmic representation for the fluorescent signals. The data were then analyzed using the FACSDiva software from Becton Dickinson. As negative controls, the yeast cells alone were labeled with anti- CD16-APC and calcein-AM, and the phagocytic cells alone were labeled with CFW and used to determine the background of each fluorescent marker. Microscopy Aliquots of phagocytosing macrophages and neutrophils were deposited onto glass slides and observed with a Zeiss Axioplan microscope. The images were recorded with a Micromax camera (Princeton Instruments). The wells of the plate were observed using a Zeiss Axiovert 200 microscope and the images were recorded with a Axiocam ICm1 camera (Zeiss). Video-microscopy 26105 phagocytes were plated in 9 cm2 Petri dishes with glass bottoms (Iwaki). 46105 yeast cells in 2 ml of cRPMI medium were added to the phagocytes to start the infection. The movies were recorded at the BIC (Bordeaux Imaging Center), using an inverted video-microscope (Leica) equipped with a QuantEM camera. A recording was done every two minutes for 6 to 9 hours at 37uC under 5% CO2, at five different positions on the plate. Z steps were carried out (2 mm step on a range of 14 mm) for each recording. The images were analyzed using the Metamorph Offline software (Molecular Devices). Statistical analysis The differences presented in the Results section were all tested for significance using ANOVA and t tests. Differences were considered significant when P values were , 0.05. Quantification of Candida - Phagocytes Interaction Supporting Information Figure S1 Yeast cells multiplication in cRPMI medium with and without CFW (5 mg/ml) by OD600nm measure- ments. C.a: C.albicans, C.g: C.glabrata, C.l: C.lusitaniae. (TIF) Figure S2 Macrophages viability in cRPMI medium with and without CFW (5 mg/ml) by calcein fluorescence measurements. Each bar is the average of two experiments 6 standard error. (TIF) Figure S3 Calcein fluorescence varies proportionaly with the number of macrophages. Each bar is the average of three experiments 6 standard error. (TIF) Figure S4 Yeast cells muliplication over time by OD600nm and CFW fluorescence measurements in cRPMI medium with 5 mg/ml of CFW. Dashed lines show linear regression lines, and their slopes are indicated. (TIF) Figure S5 Analysis of the interactions involving the J774 macrophages and stationary-phase living yeast cells at 1M:1Y MOI over 24-hour time course experiments. Figure S5 details how the diagram of Figure 3A was built. Figure S5A shows the flow cytometry analysis of the macrophages and corresponds to the left part of the diagram of Figure 3A. Each bar represents the viability of infected macrophages compared to uninfected macrophages, and the numbers indicate the parts of phagocytosing (shaded tones) or non-phagocytosing (white tones) macrophages. Note that C. albicans engaged the higher part of macrophages in phagocytosis (T 30 min) and killed more macro- phages (T 5 h and T 24 h) than C. glabrata and C. lusitaniae. Figure S5B shows the fluorometry analysis of the fungal cells and corresponds to the right part of the diagram of Figure 3A. Each bar represents the percentage of the total fungal biomass internalized in viable macrophages. Note the lower uptake of C. albicans cells. Each condition was performed in quintuplet (A) or in triplicate (B). Each bar is the average of three independent experiments 6 standard error. (TIF) Figure S6 Representative pictures of the J774 macro- phages after 5 hours of infection with the three Candida species in culture flasks at 1M:1Y MOI. Note that the totality of C. glabrata cells were engulfed, whereas C. albicans (mostly in filamentous form) and C. lusitaniae cells were still observed outside the macrophages. The scale bars represent 30 mm. See also Movie S1 showing the interaction of J774 macrophages with C. albicans at a MOI of 1M:1Y over a 5-hour incubation period. (TIF) Figure S7 Macrophage fungicidal activity toward the different Candida species. To investigate the capacity of the macrophages to kill the different species of Candida, the survival of the yeast cells was determined following 5 and 24 hours of incubation with phagocytic cells at a 1M:1Y MOI (Method S2). 100% of the C. albicans cells survived, while 20% and 40% of the C. glabrata and C. lusitaniae cells were killed within 24 hours of interaction with the macrophages, respectively. C. a: C. albicans, C. g: C. glabrata, C. l: C. lusitaniae. (TIF) Method involved in glucose assay. See Table Method S1 S1. PLoS ONE | www.plosone.org 14 March 2012 | Volume 7 | Issue 3 | e32621