Quantitation of neutrophil phagocytosis, using fluorescent latex beads. Correlation of microscopy and flow cytometry

Abstract

Phagocytic particle uptake was calculated from data obtained by microscopy and flow cytometry utilizing commercially available fluorescent beads of uniform (2 micrometer diameter) size. PMNs collected from a Ficoll-Hypaque density gradient were incubated with the fluorescent beads in human plasma and gently washed in buffer to remove most of the extracellular beads. The cells were observed and analyzed by fluorescence microscopy (sample size = 300 cells) and flow cytometry (sample size = 50,000 cells) to determine the percent phagocytic cells, number of beads per phagocytic cell, and beads per 100 PMNs. Correlation of these indices for microscopic and machine counts was in high agreement, with r values greater than 0.80. Since flow cytometry increases the sample size 500-fold above that attained by manual microscopic counts, the coefficient of variation is proportionately reduced. Unlike other automated techniques for phagocytosis such as the use of radiolabeled particles, flow cytometry yields information on the cell population such as percent of phagocytic cells and the distribution of intracellular fluorescent beads per cell as well as particle uptake. Thus the advantages of speed, greater statistical accuracy, and the ability to accumulate information on the cell population indicate that flow cytofluorometry has unique applications for the study of phagocytosis. Application of the technique to in vitro stimulation of blood neutrophil phagocytic uptake is included.