Your high MTT viability with doxorubicin is likely due to assay interference (doxorubicin’s strong color/fluorescence affects MTT readings) or biological resistance of MCF-7 (drug efflux, poor uptake, cell density effects). To resolve this, run drug-only MTT controls, confirm drug activity with an alternative assay (ATP or resazurin), and check intracellular doxorubicin uptake.

Hi Maisra Azhar,

Thank you for your answer. For the influence of doxorubicin color and fluorescence in the MTT assay, I always wash the cells with PBS after the incubation time with doxorubicin and before adding MTT. I checked the effect of doxorubicin on other cells, and the results were better. Please tell me how I can fix the poor intracellular doxorubicin uptake.

Check drug integrity and handling (fresh aliquots, light-protected); verify intracellular accumulation by measuring doxorubicin autofluorescence with flow cytometry or confocal. If uptake is low, test for P-glycoprotein–mediated efflux using a P-gp inhibitor (e.g., verapamil or tariquidar) and remeasure uptake. Probe lysosomal sequestration with a lysosomotropic agent (e.g., chloroquine) to see if nuclear delivery increases. Optimize treatment conditions (lower serum during short uptake assays, ensure cells are in log phase, test a wider concentration/time range). If biological barriers persist, consider alternative formulations (liposomal or nanoparticle DOX) or co-delivery with efflux/trafficking modulators.