A novel method using confocal laser scanning microscopy for sensitive measurement of P-glycoprotein-mediated transport activity in Caco-2 cells

Objectives The aim of this study was to use time-lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P-glycoprotein activity in Caco-2 cells. Methods The change in the fluorescence of residual rhodamine 123 at the apical and central regions of Caco-2 cells was measured in the presence of digoxin or St John's wort by using time-lapse confocal laser scanning microscopy. The data were compared with measurements made using conventional techniques, a fluorescence microplate reader and a fluorescence microscope. Key findings The percentage decrease of rhodamine 123 caused by 10 mu M digoxin or 0.1 mu g/ml St John's wort was significantly larger in the apical region of the Caco-2 cell than in the central region or in the whole cell. The digoxin-induced inhibition in the apical region as measured by time-lapse confocal laser scanning microscopy was greater than that measured in the whole cell by a microplate reader or a fluorescence microscope. Conclusions The assay of residual rhodamine 123 in the apical region of Caco-2 cells by confocal laser scanning microscopy was more sensitive than the conventional methods using a microplate reader or fluorescence microscopy. It will be a valuable screening tool for studying both the inhibition and induction of P-glycoprotein activity.