Chapter 52 On-Line Flow Cytometry: A Versatile Method for Kinetic Measurements

Publisher Summary
This chapter describes flow cytometer (FCM), which is used to perform real-time kinetic studies, including the software necessary to acquire and analyze data. It is followed by a description of two applications of on-line FCM. First, the real-time analysis of chromatin structure changes by the enzymatic digestion of the DNA is shown. Changes in the fluorescence intensity of bound DNA stains occurred within minutes. These experiments demonstrate the applicability of on-line FCM to study fast kinetic changes. Second, an assay is described for measuring the uptake kinetics of anthracycline drugs. Uptake profiles and changes therein after the addition of membrane transport modulators show the presence of typical multidrug-resistant cells. This assay is much more accurate and sensitive then classical FCM. FCM has become a highly developed cell analysis technique. Its use is widespread among a great variety of disciplines, and new areas of application are being exploited continuously. It is ideally suited for the quantitation of the uptake of virtually any fluorescent substance by cells or cell organelles. With classical FCM, quantitative analysis of the uptake of fluorescent dyes by cells has to be done by examining cell samples at intervals. This method becomes very difficult to perform when the speed of the kinetic phenomenon of interest is very fast.