Generating Chemoresistant Prostate Cancer Cells: A Procedure for Obtaining Drug-resistant Cancer Cells In Vitro
DEŞİFRE METNİ
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 5 nanomolar.
After 72 hours, aspirate the drug-containing media and add fresh docetaxel-free media. Change the media every 3 to 4 days, and wait for 1 to 2 weeks until clones appear in the flask. Aspirate the media. Carefully wash the cells with 15 milliliters of PBS and incubate them with 4 milliliters of 0.05% trypsin-EDTA for 3 to 5 minutes at 37 degrees Celsius to detach the cells from the flask surface. Then, resuspend the trypsinized cells from the flask using 8 milliliters of fresh media.
Pool the cells from all treated flasks and pellet them by centrifugation. Following that, remove the supernatant. Resuspend 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 5 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 a docetaxel dose-escalating manner and keep pooling the surviving clones after every concentration treatment. At the end of the process, you will obtain a pool of resistant cells ready for experimental use.
