A METHOD FOR STUDYING PLASMA-MEMBRANE TRANSPORT WITH INTACT-CELLS USING COMPUTERIZED FLUOROMETRY

A new method is presented for measuring rapid efflux of fluorescent co mpounds from monolayer cells, Cells grown on a glass coverslip were lo aded with a fluorescent substrate. Thereafter, the coverslip was insta lled outside the light path in a stirred and thermostated cuvette of a fluorometer, The efflux was recorded by measuring the changes of fluo rescence in the extracellular medium. The method was used to study the kinetics of active and passive plasma membrane transport of the P-gly coprotein substrates rhodamine 123 and daunorubicin, The method has ad vantages over other methods: (1) no radioactively labeled substrate is needed, (2) fluorescence of the transported substrate is not compromi sed by the cells, (3) changes in the extracellular concentration of th e substrate can be monitored continuously and therefore a substantial improvement of the kinetic resolution is obtained, and (4) the measure ment setup is relatively simple and a standard fluorometer can be used . From the efflux data, cellular transport parameters could be calcula ted, such as passive permeation coefficients and active transport rate s. (C) 1998 Academic Press.