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

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.