Kinetics of virus adsorption to single cells using fluorescent membrane probes and multiparameter flow cytometry |
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Authors: | J F Leary M F D Notter |
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Institution: | (1) Departments of Pathology and Pediatrics, University of Rochester Medical Center, 14642 Rochester, New York;(2) Department of Anatomy and Microbiology, University of Rochester Medical Center, 14642 Rochester, New York |
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Abstract: | Different genetic stains of avian RNA tumor virus (ATV) were labeled with the fluorescent membrane probe R-18 (rhodamine conjugated
to a hydrocarbon chain) and cellular receptors for virus infection were analyzed on a rapid, single-cell basis by a multiparameter
cell sorter. Chicken cells genetically susceptible to various R-18 ATV were found to adsorb much more virus, as measured by
increased fluorescent binding, than did genetically resistant chicken cells. Virus binding to receptor sites could be saturated
with increased concentrations of labeled virus. This binding could be altered by removal of the polycation, polybrene, indicating
the important influence of electrostatic forces. Correlated time measurements of virus binding to single cells were taken
with these fluorescence measurements allowing for a minute-to-minute study of the kinetics of viral adsorption to resistant
and susceptible cells. The ratio of fluorescence (proportional to the number of virions bound per cell) to light scatter (proportional
to cell surface area) on a cell-to-cell basis was analyzed to examine the heterogeneity in fluorescent virion bound per unit
cell surface area within a given cell type. With these calculations, it was found that a large amount, but not all, of observed
fluorescence heterogeneity merely reflects differences in cell surface areas. However, there are significant differences in
viral receptor site densities within this supposedly homogeneous population of cells. This study represents a successful application
of fluorescent membrane probes and flow cytometry to the study of cellular responses to viral infection at the single-cell
level. Sine large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant
cells can be cloned by multiparameter cell sorting. |
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Keywords: | Viral adsorption to single cells kinetics of avian tumor virus kinetics of adsorption to cells receptors viral on single cells binding kinetics of viruses to cells membrane viral binding to fluorescence in viral binding kinetics flow cytometry and viral binding to cells infection and kinetics of viral binding to cells cytometry viral binding kinetics by flow |
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