Figure 3. Function of efflux pump, P-gp on multicellular BBB spheroids.
(a) Immunofluorescence images showing the expression of efflux pump, P-gp (green) on the surface of the spheroid after 48 h of co-culture. HBMECs (red) were pre-labelled with CellTracker Orange before spheroid formation. Scale bar, 50 μm. (b) Fluorescence images acquired using confocal microscopy showing that increasing concentration of a P-gp inhibitor, LY335979 increases influx of rhodamine 123 (Rh123; 0.5 μg ml−1), a substrate of P-gp into the spheroids. Scale bar, 100 μm. (c) Bar graph depicting the influx of Rh123 into the spheroid with increasing LY335979 concentration. The graph shows the mean rhodamine fluorescence intensity quantified from images in b, with s.d. error bars (**P<0.01; nspheroid=11). Statistical analyses were performed using the one-way ANOVA and Tukey’s multiple comparison test.