4.5. Measurement of Doxorubicin Accumulation and Efflux

The measurement of DOX accumulation in cells was performed according to the method in [19]. Cells were seeded on microscope imaging chambers at a concentration of 2 × 10⁶ cells/mL. Then the chambers were placed on the microscope stage. To study the effect of glycoside on the time course of DOX accumulation, EAC cells were pre-exposed to different concentrations of CA₂-2 for 30 min before being mounted in the microscope stage. DOX at a final concentration of 5 μM were added to the cells and serial images at 1-min intervals were collected for 2 h and analyzed using a confocal microscope Axiovert 200M LSM510 META (Carl Zeiss, Germany). Doxorubicin fluorescence was excited with an argon laser at 488 nm, and the emission was collected through a 505-nm long-pass filter. Post-data acquisition image analysis was performed using LSM 510 software release version 4.2 (Carl Zeiss, Germany). Cell images were analyzed as mean DOX fluorescent intensity per pixel in a region of interest (nuclei). Results were obtained from analyzing images from three to five experiments.

To study of doxorubicin efflux (retention), EAC cells were seeded to a 24-well plate. Different concentrations of CA₂-2 were added to each well and plates were incubated at 37 °C for 30 min to block MDR. Then, DOX (5 μM, final concentration) was added and plates were incubated for 2 h at 37 °C to accumulate doxorubicin in the cells. After a certain time, aliquots of 100 μL of the cell suspension were transferred into a 1.5 mL Eppendorf tube, pelleted by centrifugation, and washed twice with cold PBS. Then 100 μL of cell suspension was replaced with a black 96-well microplate and the fluorescence of doxorubicin was measured using a Fluoroscan Accent (Finland) plate reader at λex = 485 nm and λem = 620 nm.