Simultaneous cytofluorometric measurement of phagocytosis, burst production and killing of human phagocytes using Candida albicans and Staphylococcusaureus as target organisms

Objective Polymorphonuclear leukocytes (PMN) play a central role in the eli mination of most extracellular pathogens, and an impairment of their functi ons predisposes an individual towards local. and systemic bacterial and fun gal infections. Here we describe a rapid and easy-to-perform cytofluorometr ic assay for investigation of PMN activity using Candida albicans and Staph ylococcus aureus as target organisms. Methods Phagocytes were stained with anti-CD13-RPE antibody, and microorgan isms were stained with calcein-AM. Oxidative burst production was measured by oxidation of dihydroethidium. The percentage of killed target organisms after ingestion was determined by staining with ethidium-homodimer-1 after lysis of human cells. The dyes and procedures used in this method were chos en after comparison of different stains and cell preparation techniques des cribed in previous assays. Results Concerning phagocytosis, the percentages of active phagocytes and o f ingested microorganisms were determined. Furthermore, the method allowed measurement of the resulting percentage of PMNs producing respiratory burst , and of the percentage of killed microorganisms. We minimized artifactual changes, which might have been the reason for the difficulties and conflict ing results of other cytofluorometric methods. Conclusions The described method provides a new whole blood cytofluorometri c assay, which combines rapid and simple handling with high reproducibility of results obtained by investigation of PMN activity using Candida albican s and Staphylococcus aureus as target organisms.