The overall goal of this protocol is to provide a simple method to generate and isolate cell cycle-arrested cells with complex karyotypes. This method can provide answers to key questions in the field of aneuploidy research, such as the interaction of the immune system with aneuploid cells. The main advantage of this technique is that it generates a cell population that harbors only complex karyotypes.
To begin the synchronization of RPE-1 hTERT cells, use freshly thawed cells and dispense them into a 10 centimeter tissue culture dish using eight milliliters of standard growth medium. Next, use a hemocytometer to determine the cell number. Then, incubate the cells overnight.
The following day, replace the standard growth medium with medium containing five millimolar thymidine. Incubate for 24 hours at 37 degrees Celsius to synchronize cells at the G1S border. After 24 hours, wash the cells three times with PBS.
Then, add standard growth medium to the washed cells and incubate for six hours at 37 degrees Celsius. First, wash the cells with 10 milliliters of PBS. Then, add medium containing 500 nanomolar Mps1 Inhibitor reversine.
Incubate for 12 hours at 37 degrees Celsius. The following day, wash the cells three times with PBS. Then, add standard growth medium and return the plates to the incubator.
Approximately 72 hours after the first aberrant mitosis, wash the cells with 10 milliliters of PBS. Then, add medium containing 330 nanomolar nocodazole. Incubate for 12 hours at 37 degrees Celsius.
The next day, aspirate the nocodazole medium. Then add three milliliters of PBS to the dish and tap the side of the plate to shake-off the mitotic cells. Rotate the plate between each tap to allow even removal of mitotic cells.
After the initial shake-off, aspirate any liquid adhered to the lid and edge of the plate. Then, check the plate under the microscope at 10X magnification. Then wash the cells with five milliliters of PBS.
Next, add medium containing 330 nanomolar nocodazole. Incubate for 12 hours at 37 degrees Celsius. After the final shake-off, add 10 milliliters of standard growth medium.
Incubate the cells for 12 to 24 hours at 37 degrees Celsius before performing the assays. The cells remaining on the plate are referred to as arrested with complex karyotype or ArCK cells. This protocol describes a simple method to isolate ArCK cells and several features specific to ArCK cells are analyzed to confirm their isolation.
ArCK cells show increased levels of cell cycle inhibitors p53, p21, and p16 in the immunoblot analysis, while euploid and aneuploid cycling cells do not. Additionally, ArCK cells are positive for senescence associated beta-Galactosidase, which stains blue-green while euploid control cells are not. Segmentation plots reveal that ArCK cells are aneuploid, characterized by multiple, random chromosome gains and chromosome losses.
Segmentation plots show the copy number of single cells from chromosome one to X, relative to a euploid reference log two scale. While attempting this procedure, it's important to make sure to remove all mitotic cells during each shake-off. After performing this procedure, other methods such as cytokine assays can be performed.
These assays will help in answering additional questions such as the ability of ArCK cells to interact with the cells of the immune system. Don't forget that nocodazole is hazardous. Appropriate personal protective equipment should be worn while using this reagent.
Once mastered, this technique can be used to efficiently isolate ArCK cells in vitro. This procedure can be done in seven days if performed properly.
