This study investigates the survival of drug-resistant cancer cells in a heterogeneous tumor cell population following treatment with an anti-cancer drug. The methodology involves seeding cells, applying drug treatments, and using crystal violet staining to identify and quantify drug-resistant colonies.
In a heterogeneous tumor cell population, treatment with an anti-cancer drug would kill most cells, but drug-resistant cells survive, resulting in therapeutic failure.
To determine the proportion of drug-resistant cells, seed a heterogeneous tumor cell population in a multi-well plate. Incubate to facilitate cell adherence. Add an increasing concentration of a suitable anticancer drug solution.
Interaction with drug molecules causes sensitive cells to die and, subsequently, detach from the culture plates. In contrast, drug-resistant cells survive and remain attached to the plate.
Aspirate the spent media to remove the detached cells. Add fresh drug-free media and incubate the plate under physiological conditions. The drug-resistant cells proliferate and form tumor colonies. Now, add crystal violet staining solution to the colonies and incubate.
Crystal violet, a purple-colored, membrane-permeable, positively-charged stain enters the cells and binds anionic proteins and DNA, thereby imparting a purple color to the cells.
Next, add ethanol to solubilize the unreacted stain. Wash to remove any excess stain and air-dry the plate.
Drug-resistant colonies appear as purple dots. Count the number of colored colonies in each well. Represent the percentage of cell viability on a graph to determine the proportion of drug-resistant cancer cells at each drug concentration.
In this procedure, plate 2000 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 one to two 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 two to three 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.
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