4.7. Rhodamine 123 Accumulation Assay

We conducted an investigation using flow cytometry to examine the function of P-gp, a protein that transports substances out of cells. Specifically, we wanted to see how the EC and ES extracts affected the accumulation of the P-gp substrate rhodamine 123 (Rho123) [173] in two types of P-gp-overexpressing cells (NCI-H460/R and U87-TxR) and compared the results with those from control cells (NCI-H460 and U87). To carry out the experiment, we grew all cell lines to 80% confluence in 25 cm² flasks, collected the cells, and put them in a solution containing Rho123 (2.5 µmol L⁻¹). We immediately treated the MDR cells with the EC and ES extracts (20 µg mL⁻¹, the average IC50 calculated for all tested cancer cell lines) and incubated them at 37 °C in 5% CO₂ for 30 min. After the accumulation period, we washed the samples twice, collected the cells, and analyzed them using a CytoFLEX flow cytometer (Beckman Coulter, IN, USA). The orange fluorescence of Rho123 was measured on fluorescence channel 1 (FL1) at 525 nm. We tested at least 20,000 events for each sample, and the mean fluorescence intensities were analyzed using Summit v4.3 software (Dako Colorado Inc., Fort Collins, CO, USA). We analyzed the mean ± SEM values from three independent experiments using GraphPad Prism 8 (San Diego, CA, USA) and used Sidak’s multiple comparison test for two-way ANOVA for the statistical analysis.