Leary J F, Notter M F
Cell Biophys. 1982 Mar;4(1):63-76. doi: 10.1007/BF02788556.
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. Since large numbers of cells can be examined rapidly, small subpopulations of live virally susceptible or resistant cells can be cloned by multiparameter cell sorting.
用荧光膜探针R - 18（与烃链共轭的罗丹明）标记禽RNA肿瘤病毒（ATV）的不同基因株，并通过多参数细胞分选仪在快速单细胞基础上分析病毒感染的细胞受体。通过增加的荧光结合来测量，发现对各种R - 18 ATV基因敏感的鸡细胞比基因抗性的鸡细胞吸附更多的病毒。标记病毒浓度增加时，病毒与受体位点的结合可达到饱和。去除聚阳离子多聚赖氨酸可改变这种结合，表明静电力的重要影响。这些荧光测量与病毒与单细胞结合的相关时间测量同时进行，从而能够对病毒吸附到抗性和敏感细胞的动力学进行逐分钟研究。分析细胞间荧光（与每个细胞结合的病毒粒子数量成正比）与光散射（与细胞表面积成正比）的比率，以检查给定细胞类型内每单位细胞表面积结合的荧光病毒粒子的异质性。通过这些计算发现，观察到的大量但并非全部荧光异质性仅仅反映了细胞表面积的差异。然而，在这个假定均匀的细胞群体中，病毒受体位点密度存在显著差异。这项研究代表了荧光膜探针和流式细胞术在单细胞水平研究细胞对病毒感染反应中的成功应用。由于可以快速检查大量细胞，通过多参数细胞分选可以克隆少量存活的病毒敏感或抗性细胞亚群。
