Rhodamine 123 Uptake Assay

Rhodamine uptake/efflux assays were conducted as described by Chieli et al. (1993) with minor modifications. After five passages in media containing vincristine (50 μM), Caco-2 VCR (Eneroth et al., 2001) cells (1 × 10⁴ cells/well) were incubated for 2 h with the P-gp inhibitor verapamil (20 μM) or plant extracts (100 μg/mL) in serum-free media containing rhodamine 123 (5 μg/mL). After incubation, cells were washed with verapamil (20 μM) in PBS. Cells were lysed with 100 μL of 0.1% Triton X-100 in PBS and the plates were placed in the incubator for 15 min. The fluorescence intensity of cell lysates was measured in the plate reader (Exc-485 nm, Em-525 nm). The cellular accumulation of rhodamine 123 for each of the extracts was expressed as the percentage of the accumulation measured for rhodamine 123 under control conditions.