Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis

Abstract

A simple flow cytometric method (FCM) for measuring phagocytosis of Staphylococcus aureus by human neutrophils (polymorphonuclear leukocytes [PMNs]) is described. This assay utilizes 100 microliters of EDTA-anticoagulated whole blood and a simplified method of fluorescently labeling bacteria. A commercially available whole-blood lysing reagent allows for the removal of erythrocytes and the exclusion of external free or adherent bacteria. Phagocytized bacteria are unaffected by this reagent, so PMNs containing internalized bacteria can be easily identified by FCM. Advantages of this method include the following: (i) small sample size, (ii) no requirement for PMN separation, (iii) rapid reliable method of labeling the bacteria, (iv) ability to distinguish between adherent bacteria and those which are actually internalized, (v) avoidance of vital dyes as quenching agents, and (vi) ability to fix cells and store for future FCM analysis.