2.8. Rhodamine 123 efflux assay

Cells (4 × 10⁵) grown in a six‐well culture dish for 6 hours before respective treatments (like‐17AAG and quercetin for 24 hours) and were washed with PBS, incubated with 1 μM concentration of Rhodamine 123 (Rh123, #R302, Thermo Fisher Scientifics) in the incomplete medium at 37°C for 30 minutes. After the incubation, cells were washed with PBS, and the incomplete medium was added, another set was incubated with 5 μM of cyclosporine A (#30024‐25MG, Sigma‐Aldrich) for 2 hours to examine the maximum drug accumulation. Cells were washed with 1× PBS and scraped into 1 mL of PBS on ice and analyzed using a flow cytometer (Beckman Coulter, Gallios). The efflux ratio was calculated by the mean channel value of Cyclosporin A/mean channel value of treatments as per our previous publication. ²³