A single-step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane-bound and ingested Candida albicans in phagocytosing neutrophils

Background: Distinguishing ingested particles from those attached to the cell Surface is,in essential requirement when performing quantitative studies of plagocytosis. In the present report, we describe a simple, sensitive and reliable flow cytofluorometric method that achieves this goal in a Candida albicans-human neutrophils (PMN) system. Methods: The assay is based on the observation that the vital dye trypan blue (TB), while quenching the green fluorescence of fluorescein-labeled C albicans, causes them to fluoresce red. PMN were incubated with fluorescein-labeled yeast particles for the required time. Aliquots of the incubation mixtures were then promptly diluted with an equal volume of a TB solution at pH 4.0, and subsequently analyzed by flow cytometry for green and red fluorescence. Results: Since TB does not penetrate into the cells, ingested yeasts retain their green fluorescence, while membrane-bound particles display a red fluorescence. Conclusions: Our fluorescence flow cytometric method enables to simultaneously distinguish, within the leukocyte population, cell subsets with attached and ingested yeast particles. Its major features are: (1) accuracy, sensitivity and reproducibility; (2) no further sample manipulations after completion of phagocytosis; (3) possibility of counting free, attached and internalized yeast particles; and (4) use of a nontoxic reagent (TB). (C) 2004 Wiley Liss, Inc.