Molecular mechanisms of neutrophil aggregation measured by flow cytometry

S. I. Simon, L. A. Sklar

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Abstract

Flow cytometry has enabled measurement of the kinetics of formyl peptide stimulated neutrophil aggregation and its dependence on the CD11b/CD18 adhesion molecule (Simon et al., 1990; JCB:111). We are currently measuring aggregation of neutrophils in whole blood using flow cytometry. Fresh whole blood samples were kept at 4°C and stained with LDS-751 a vital nucleic stain. Cytometric detection of neutrophil aggregation in whole blood and isolated cells at 37°C was achieved by thresholding on LDS-751 fluorescence and then gating on forward and right angle light scatter. Aggregation was up to 5 times more efficient in whole blood than in purified cells, despite the fact that the number of CD11b/CD18 sites was upregulated 5-10 fold in clutriated neutrophil preparations. The time course of whole blood aggregation was often irreversible as compared to isolated cells. Aggregation was essentially blocked by preincubation with saturating concentrations of antibody to the CD18 integrin. CD11b-CD18 enables transient cell-cell adhesion as a function of their number and lifetime within the contact region between cells. An estimate of the number of sites in the contact region was made by comparing the ratio of fluorescent probe on aggregates and singlets for both CD18 fluorescen conjugated monoclonal antibody Fab fragments and control fluorescent probes. Cytometric data was collected on the peak, integral, and time of flight (width) of the CD18 fluorescent pulse. It was estimated that no more than 10-15% of the CD18 labeled adhesive sites were confined to the contact region on adjacent cells, where they are inaccessible to the CD18 antibody. We are currently examining the activity, number, and topography of CD11b/CD18 adhesive sites on the kinetics and efficiency of neutrophil collisions in whole blood and isolated cells.

Original languageEnglish (US)
Pages (from-to)603-604
Number of pages2
JournalAnnals of Biomedical Engineering
Volume19
Issue number5
Publication statusPublished - 1991
Externally publishedYes

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ASJC Scopus subject areas

  • Biomedical Engineering

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