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

1. Functional Characterization of Acquired Docetaxel Resistance by Colony Formation Assay

NOTE: In this protocol, chemoresistance has been evaluated using colony formation assays. Alternative methods used to evaluate cell viability (i.e., MTS assays) can be used based on investigators' preferences.

1. Plate 2,000 cells (DU145 or 22Rv1, both parental and Docetaxel-resistant) per well in 6-well plates, using 2 mL of media per well.
2. After 24 h, add increasing concentrations of Docetaxel (parental cells: 0.25, 0.5, 1, 2.5 and 5 nM; DR cells: 50, 125, 250, 500 and 1,000 nM). 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.
3. After 72 h, aspirate the drug-containing media and add fresh Docetaxel-free media.
4. Incubate plates for 1-2 weeks until colonies are visible under the microscope.
5. To stain the colonies, wash them gently with 2-3 mL PBS and incubate with 2-3 mL crystal violet solution (0.1% w/v in 10% formalin) for 20 min inside the tissue culture hood or a fume hood.
6. Remove staining solution, wash plates with 2-3 mL of H₂O, remove H₂O and air-dry plates.
7. Analyze the result by visualizing the wells and manually counting colonies with the help of a marker pen (to avoid counting the same colonies twice) and represent the percentage of cell viability in a graph. Take digital images of the plates for figure representation (Figure 1A).