The overall goal of this procedure is to generate Docetaxel-resistant cancer cell experimental models as a platform to study molecular mechanisms driving anti-mitotic therapy resistance. This method can help answer key questions in the cancer cell biology and genomateic fields, such as identifying the signaling pathways contributing to disease progression, and determining potential targetable strategies. The main advantage of this technique is that it is a reliable and easy way to generate experimental models to study anti-cancer drug responses besides using patient tumor samples, and animal models.
Demonstrating the procedure will be Lisa Mohr, a postdoc from my laboratory, and Jungreem Woo, another postdoc on our team. To begin this procedure, plate DU145 or 22Rv1 cells in the 150 centimeter squared flasks containing 20 milliliters of media. After 24 hours, when the cells are at about 70 to 80%confluence, add Docetaxel at five nanomolar.
After 72 hours, aspirate the drug containing media, and add fresh Docetaxel-free media. Change the media every three to four days. Wait for one to two weeks until clones appear in the flask.
Aspirate the media, carefully wash the cells with 15 milliliters of PBS and incubate them with four milliliters of 0.05%Trypsin-EDTA for three to five minutes at 37 degrees Celsius to detach the cells from the flask surface. Then, re-suspend the Trypsinized cells from the flask using eight milliliters of fresh media. Pull the cells from all treated flasks and pellet them by centrifugation.
Following that, remove the superna-den, re-suspend the cell pellet in 20 milliliters of fresh media, and plate the cells in 150 centimeter squared flasks. After 24 hours, when the cells are at about 70 to 80%confluence, add five nanomolar Docetaxel again. Repeat the procedures as shown previously until the clones appear in the flask.
Then plate the cells in 150 centimeter squared flasks again. After 24 hours, when the cells are at about 70 to 80%confluence, treat them with 10 nanomolar Docetaxel. Repeat the same steps in the Docetaxel dose escalating manner, and keep pooling the surviving clones after every concentration treatment.
At the end of the process, you'll obtain a pool of resistant cells ready for experimental use. In this procedure, plate 2, 000 cells using 2 milliliters of media per well in the six 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 two to three milliliters of PBS. 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 standing solution and wash the plates with two to three 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. Here is a diagram depicting the determination of the Docetaxel IC50 in the parental prostate cancer cell lines DU145 and 22Rv1. The expected percentages of cell viability in the Docetaxel dose escalating concentrations needed are shown here.
And here are the representative Brightfield microscopy images of DU145 and 22Rv1 cells at the indicated times during the generation of Docetaxel resistance. Red arrows indicate a Docetaxel-resistant clone after the protocol was completed. Shown here are the representative colony formation assays corresponding to the Docetaxel-resistant cells functional validation.
Cells were exposed to increasing Docetaxel concentrations and survival evaluated by crystal violet staining. After watching this video, you should have a good understanding of how to generate and validate drug-resistant cell lines, which can then be used for examinations and procedures of your choice such as gene expression analysis and genetic manipulations. Thanks for watching and good luck with your experiments.
