Quantification of Candida - Phagocytes Interaction Figure 7. Analysis of human neutrophil interaction with live yeast cells in stationary phase at different MOIs over 5-hour time course experiments. The diagram shows the flow cytometer analysis of the neutrophils. The horizontal bar represents the neutrophil survival, indicated as a percentage on the left side of the bar. The white part of the bar represents the percentage of non-phagocytosing neutrophils, the shaded tones part represents the percentage of phagocytosing neutrophils. Note that more neutrophils died when infected with C. albicans than with C. glabrata and C. lusitaniae. Each condition was performed in quintuplet per experiment. Each bar is the average of three independent experiments 6 standard error. doi:10.1371/journal.pone.0032621.g007 process to allow the continuous labeling of newly replicated yeasts. An increase in CFW fluorescence over time was interpreted as an increase in the fungal biomass, independently of the morphology and size of the cells. As the CFW can not enter viable macrophages, the multiplication of yeasts within macrophages was not assessed, unless yeasts were released in the medium. Phagocytes were specifically labeled with calcein-AM and anti- CD16-APC antibodies. Calcein-AM is a fluorogenic esterase substrate, used as a viability probe that reflects both enzymatic activity, which is required to convert calcein-AM to a fluorescent product, and cellular membrane integrity, which is required for intracellular retention of the fluorescence. Calcein-AM was chosen because cell viability studies showed it was quite well retained by living cells and released during cytolysis, with low pH sensitivity [23]. Anti-CD16-APC antibodies specifically labeled the mem- brane of phagocytes. To avoid the counting with the microscope, we chose flow cytometry to analyse the population of phagocytes. This method allows a rapid analysis of a large number of cells, and a simultaneous measure of two parameters at the individual cell PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e32621