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

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.