Functional Chemoresistance Characterization: A Method to Evaluate Drug Resistance in Cancer Cells Using Clonogenic Assay

JoVE Encyclopedia of Experiments

Kankeronderzoek

0 weergaven • 3:28 min • April 30th, 2023

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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.

In this procedure, plate 2,000 cells using 2 milliliters of media per well in the 6-well plates. After 24 hours, add increasing concentrations of docetaxel for both DU145 and 22Rv1 cell lines. Add DMSO only to one well as a control at the same volume used for the highest docetaxel dose. After 72 hours, aspirate the drug-containing media and add fresh docetaxel-free media.

Incubate the plates for 1 to 2 weeks until colonies are visible under the microscope. To stain the colonies, wash them gently with 2 to 3 milliliters of PBS and incubate them with 2 to 3 milliliters of crystal violet solution for 20 minutes inside the tissue culture hood or a fume hood.

Afterward, remove the staining solution and wash the plates with 2 to 3 milliliters of water. Then, remove water and air-dry the plates. Take digital images of the plates for figure representation. Analyze the result by visualizing the wells and manually counting the colonies with the help of a marker pen and represent the percentage of cell viability in a graph.

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Laatst bijgewerkt: 15 augustus 2026