Functional Chemoresistance Characterization: A Method to Evaluate Drug Resistance in Cancer Cells Using Clonogenic Assay
Transcrição
To evaluate chemoresistance in cancer cells, begin by taking a multi-well plate. Seed a suspension of the desired drug-sensitive prostate cancer cells into a few wells and a suspension of the drug-resistant variant of the cells into the remaining wells.
Incubate the culture to facilitate the attachment of cells to the culture plate. Treat both the cell types with a range of increasing doses of the selected drug solution. The drug molecules enter the sensitive cells and cause cell death even at their lowest concentrations. However, the resistant cells expel the drug molecules through specialized drug-efflux pumps, enabling their survival despite being exposed to higher drug concentrations.
Aspirate the spent media containing debris and drug solution. Supplement with fresh drug-free growth media. Incubate to allow the surviving drug-resistant cells to proliferate and form colonies. Add a suitable staining solution to color the live-cell colonies.
Wash the culture to remove excess dye. Count the number of colored colonies in each well. The wells containing drug-sensitive cells hold very few viable colonies. In contrast, the wells containing drug-resistant cells exhibit a higher number of colonies. However, these drug-resistant cells display a reduction in the number of colonies with increasing drug doses.
