Radiation therapy is most frequently delivered using linear accelerators. This protocol allows researchers to assess the biological effects of radiation on cells using a linear accelerator. The advantage of this technique is that by using a linear accelerator, researchers can study a wide range of clinically relevant doses and dose rates.
Furthermore, this protocol can be used for all types of cancer cells. This method can be performed using similar equipment to that shown here. For example, linear accelerators manufactured by other vendors.
Individuals not experienced in performing dosimetric calculations using a linear accelerator may struggle to calculate the correct number of monitor units required to deliver the desired dose to the sample. They would benefit by seeking advice from an experience medical physicist or dosimetrist. Delivering dose to the sample accurately using a linear accelerator requires careful placement.
Two days before scheduled irradiation, working in a culture hood, use a sterile five milliliter pipette to collect glioma stem-like cells from a culture plate into a 15 milliliter centrifuge tube. Centrifuge the cells at 200 times g for three minutes in a countertop centrifuge. Discard the supernatant and resuspend the cell pellet with one milliliter of trypsin-EDTA.
Incubate at room temperature for five minutes to digest the pellet and make a single cell suspension in trypsin-EDTA. Every two minutes during digestion, gently shake the bottom of the centrifuge tube to make sure the cells are thoroughly digested. Then add three milliliters of stem cell culture media to quench trypsin, gently mix, and centrifuge at 200 times g for three minutes in a countertop centrifuge.
After discarding the supernatant, resuspend the cell pellet with five milliliters of cell culture media and count the cells with a hemocytometer. Plate five million cells divided in two 10 centimeter plates containing 10 milliliters of cell culture media and incubate 48 hours at 37 degrees Celsius. Right before the scheduled irradiation, collect the cells and centrifuge as done before.
Discard the supernatant and resuspend the cell pellet with five milliliters of cell culture media. Transfer one milliliter of cell suspension into a 35 millimeter plate containing two milliliters of cell culture medium, making sure that the medium reaches a height of one centimeter. Transfer the plated cells to a secondary container to reduce the risk of contamination and bring the cells in the container on a utility cart to the irradiation facility to irradiate the cells.
One day before irradiation, working in the cell culture hood, use a Pasteur pipette connected to a vacuum to remove DMEM medium from the cell culture plate to the attached cell line. Then wash the cells with five milliliters of sterile PBS to rinse off residual medium. Add one milliliter of trypsin-EDTA into the culture dish and gently tilt the culture plate to make sure that the entire dish is covered.
After incubating at room temperature for five minutes, quench the trypsin-EDTA reaction with three milliliters of DMEM media. Use a five milliliter pipette to collect the cells into 15 milliliter centrifuge tube, centrifuge, and then culture the cells as described in the manuscript. To irradiate these previously prepared cells using the LINAC's console software, set the accelerator gantry and collimator to zero degrees, open the x and y jaws to a symmetrical five by five square centimeters field size, and retract the multileaf collimators.
Add at least five centimeters of water-equivalent material on the treatment couch and place the cell dish to be irradiated onto it at the center of the LINAC crosshairs. Place the cells at a depth of maximum dose in a six megavolt X-ray beam around 1.5 centimeters and add an additional one centimeter of water-equivalent material on top of the dish. Affix the front pointer to the head of the LINAC and extend it until it touches the surface of the buildup material.
Adjust the table height until the distance from the source to the buildup surface is 100 centimeters. Leave the treatment vault, making sure that all other individuals have left. Verify that there are no other cells in the room, and then close the vault door.
Confirm the field size, monitor units, and monitor units per minute at the console, and then enable the beam for a cell of radiation. Three samples of glioma stem-like cells were prepared using this protocol and collected 24 hours after irradiation. There cell cycle profiles were calculated and shown with the percentages of cells in different phases of the cell cycle.
G2 cell cycle arrest was observed after irradiation of glioma stem cells with either a standard dose rate or an extra high dose rate, compared with non-irradiated control cells. In order to have the right dose delivered, it is most important to measure the height of the media in the culture dish to make sure it reaches one centimeter. Following this procedure, other biological assays can be performed, such as immunostaining, western blot, and cell cycle analysis.
Since this procedure provides the researchers clinically relevant dose and dose rate settings, it can be used to evaluate the radiation effect on many cell culture based models.
