Figure 1. Trypan blue efficiently quenched CFW-labeled yeast fluorescence. (A) Emission spectrum of CFW-labeled yeasts before and after trypan blue (TB) quenching. 16107 C. lusitaniae cells were labeled with 5 mg/ml CFW, and 250 mg/ml trypan blue was used for quenching. Excitation wavelength was set up at 360 nm and emission wavelength was set up to cover the entire emission spectra of CFW. (B) Macrophages after 4 hours of infection with C. lusitaniae. Yeast cells were stained with CFW (blue), macrophages were stained with calcein (green). Left panel: phase contrast, right panel: fluorescence. The fungal cells ingested inside the macrophages were protected from trypan blue quenching and were fluorescent, whereas membrane-bound yeasts exposed to trypan blue quenching were not (some examples are shown with white arrowheads). A pseudohyphae of C. lusitaniae can be seen within the macrophage (black arrowhead). The bar represents 20 mm. (C) Fluorometric detection of CFW-labeled C. lusitaniae cells before and after trypan blue quenching. Trypan blue used at 250 mg/ml quenched the CFW fluorescence to background level for up to 16107 C. lusitaniae cells. doi:10.1371/journal.pone.0032621.g001 exponential phase. The J774 macrophages were infected with C. albicans, C. lusitaniae and C. glabrata blastospores from either the exponential or stationary phase at a MOI of 1M:1Y and the phenotypes of the interactions were compared over time. Results are represented in Figure 3 (see also Figure S5), showing in parallel Quantification of Candida - Phagocytes Interaction the behavior of the macrophages (percentage of viable macro- phages and percentage of phagocytosing macrophages), and the behavior of the yeast cells (increase of the total fungal biomass and percentage of engulfed fungal biomass) during the infection process. After 30 min of incubation, nearly 100% of the macrophages infected with the different yeast species were still alive. Occasionally, we observed a slight increase of the number of active macrophages when infected macrophages were compared to uninfected controls, probably due to an increased metabolism in response to the presence of yeasts. Overall, few macrophages were engaged in phagocytosis of yeast cells as early as 30 min (6 to 24% according to the yeast species). C. albicans appeared to be the more aggressive species in terms of macrophage killing (Figure 3A and 3B, left bars): only 13% of the macrophages survived after five hours of infection with stationary yeast cells; interestingly, macrophages survival was higher at T 5 h with exponential yeast cells. However, nearly all macrophages were completely destroyed after 24 h of incubation. The process of killing was dependent upon the formation of hyphae by C. albicans that pierced the majority of the macrophages (Figure S6, Movie S1). On the other hand, C. albicans was less internalized by the macrophages than the two other species (Figure 3A and 3B, right bars): only 7 to 17% of the fungal biomass was engulfed according to the duration of incubation. No fungal cells were detected in viable macrophages after the fluorescence quenching experiment, indicating that the entire C. albicans population was unengulfed at T 24 h. Thus, the majority of the cells multiplied outside macrophages, as verified with the microscope (Figure S6), leading to an important fungal biomass development (x 7 or x 4 at T 24 h). Survival of macrophages was much higher when infected with C. glabrata (nearly 80% survival at T 5 h and T 24 h), with up to 40 to 50% macrophages involved in phagocytosis (Figure 3A and 3B, left bars). There was no difference in macrophage killing between stationary and exponential yeasts. The proportion of macrophages engaged in phagocytosis with C. glabrata, as well as the proportion of viable macrophages, were similar at both T 5 h and T 24 h, indicating that C. glabrata did not kill the macrophages and that the proportion of macrophages engaged in phagocytosis reached a plateau. The rate of yeasts engulfed was greater for C. glabrata than for C. albicans (Figure 3A and B, right bars). The proportion of the C. glabrata yeasts whose fluorescence was unquenched (78%, Figure 3A, T 5 h, right bar) suggested that the vast majority of the cells were engulfed by intact macrophages. Using a microscope, we confirmed that all of the C. glabrata cells derived from stationary phase were cleared out from the medium by the J774 macrophages at T 5 h (Figure S6). The rate measured with fluorometry did not reach 100% probably because some killed macrophages allowed a portion of the engulfed fungal cells to be quenched. Candida glabrata yeast cells remained intra- macrophagic at 24 hours post-infection as indicated by the observation with the microscope and by the fact that the total CFW fluorescence did not increase between T 5 h and T 24 h. The CFW can not enter viable macrophages [18], accordingly, it was not possible to determine if the fungal cells multiplied inside the J774 macrophages. The multiplication of the C. glabrata fungal biomass was restricted (x 1.3) when stationary yeast cells were used, and was slightly higher when exponential yeast cells were used (up to x 2.3 at T 24 h). We could verify with the microscope that this increase in fungal biomass was derived from the extra-macrophagic multiplication of unengulfed yeasts, whereas the stationary yeast cells were kept fully ingested by the phagocytes (Figure S6). Furthermore, when using C. glabrata yeasts taken from exponential PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e32621