Crystal Violet Staining: A Technique to Visualize Drug-Resistant Cancer Cells Using Colony Formation Assays

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.