Flow cytometry measurement of Saccharomyces cerevisiae phagocytosis by neutrophils in mouse blood

Anotacija

We used the flow cytometric method for measuring the phagocytosis of Saccharomyces cerevisiae (S. cerevisiae) cells by mouse neutrophils. This method allows us to identify different cell populations by their light scattering properties. The heterogeneity of the mouse blood cell population was determined by forward scatter (FSC) and side scatter (SSC) cytometric profile which showed two distinct cell populations. The experimental conditions were selected in such a way that it was possible to analyse the phagocytosing function of neutrophils in pheripheral blood without time-consuming cell separation. Only 100 μl of heparinized whole blood samples was required for this determination. The phagocytosis was not dependent on the opsonins at 37 °C for 30 min. This method allowed to differentiate between the cells adherent to neutrophils and the ingested yeast cells by quenching fluorescein-labeled extracellular yeast cells with ethidium bromide. Trypan blue did not alter in fluorescence quenching the effect on ingested and adherent yeast cells. In addition, the results of this study show that cytochalasin D by 45– 46% inhibited the phagocytosis of ingested S. cerevisiae cells by mouse neutrophils. These data offer a sensitive technique which should be very useful in the further studies and estimation of the interactions between normal phagocytes and other microorganisms in pheripheral blood. Keywords: flow cytometry, neutrophils, phagocytosis, Saccharomyces cerevisiae