Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

• Published in: MICROBIOLOGY and IMMUNOLOGY 1999/06/20, Vol.43(6), pp.505-512
• Main Authors: Heinzelmann, Michael, Gardner, Sarah Appel, Mercer-Jones, Mark, Roll, Amy J., Polk Jr, Hiram C.
• Format: Article
• Language: English
• Subjects: Bacterial Adhesion; Biological and medical sciences; Evaluation Studies as Topic; Flow cytometry; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunobiology; Kinetics; Klebsiella pneumoniae - immunology; Microscopy, Fluorescence; Myeloid cells: ontogeny, maturation, markers, receptors; Neutrophils - immunology; Phagocytosis; Polynuclears; Quantitative analysis; Staphylococcus aureus - immunology
• ISSN: 0385-5600; 1348-0421
• DOI: 10.1111/j.1348-0421.1999.tb02435.x

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1 : 1, 1 : 10, 1 : 100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.