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 an essential requirement when performing quantitative studies of phagocytosis. 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).
Busetto
Sara
S
Department of Physiology and Pathology, University of Trieste, Trieste, Italy.
Trevisan
Elisa
E
Patriarca
Pierluigi
P
Menegazzi
Renzo
R
D002176:Candida albicans / Q000302:isolation & purification*; D002462:Cell Membrane / Q000382:microbiology*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D009504:Neutrophils / Q000382:microbiology*; D010587:Phagocytosis; D014343:Trypan Blue
D014343:Trypan Blue