Rhodamine 123 Uptake Assay

Rhodamine uptake/efflux assays were conducted as previously described (Chieli et al., 2009) with minor modifications. Cells were seeded into 96-well plates for 24 h to allow for attachment. Then, the growth media was changed to serum free media. Rhodamine (5 µg/ml) was added to the wells containing 100 µg/ml of all the extracts to be tested. 20 µM verapamil was used as positive control. After 2 h incubation, cells were washed with 20 µM verapamil 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. 80 µl of each of the cell lysates in each well was transferred to a 96-well black plate and the fluorescence intensity of each well was measured in a Tecan^® plate reader (Exc-485 nm, Em-525 nm). The cellular accumulation of Rh-123 for each of the extracts was expressed as the percentage of the accumulation measured for rhodamine only, that is under control conditions.