One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

The overall goal of this assay is to evaluate the major modes of clonogenic cell deaths induced by ionizing radiation based on the characteristic morphologies of nuclei stained with DAPI. This method can help answer key questions in the field of radiation biology. The main advantage of this rapid and cost-efficient technique is that the major modes of clonogenic cell death induced by ionizing radiation can be evaluated simultaneously.

Generally, individuals new to radiation biology will struggle to evaluate radiation induced clonogenic cell death because different assays are required to evaluate each mode of cell death. We first had the idea for this method when we performed large scale screening of radiation induced clonogenic cell death using multiple cell lines and radiation doses. Although this method can provide insight into radiation biology, it can also be applied to other systems, such as chemotherapy for cancer.

Demonstrating the procedure will be our collaborator, Atsushi Shibata. Start by sterilizing a scalpel with paper towels moistened with 70%ethanol. Then use the scalpel to place a cover slip in a 35 millimeter cell culture dish.

To the culture dish with the cover slip, add one milliliter of culture medium. Now gently hold the center of the cover slip with sterile forceps and aspirate the culture medium from the dish completely. Next, prepare a three milliliter cell suspension in culture medium in a 15 milliliter tube.

Add the cell suspension to the culture dish. And incubate it overnight at 37 degrees Celsius and 5%CO2. Ensure that cells are attached to the cover slip and are alive by examining them under an inverted phase-contrast microscope.

Now irradiate the dish with six grays X-ray radiation. After irradiation, incubate the cells in an incubator for 72 hours at 37 degrees Celsius and 5%CO2. Now take the cells from the incubator and aspirate the culture medium from the culture dish.

Cut the tip of a 1, 000 microliter micro pipette tip about five millimeters from the end using scissors. And use it to add one milliliter of fixation solution to the culture dish. Apply the fixation solution along the walls of the culture dish to minimize damage to the cells.

Then transfer this culture dish into a square culture dish and shake it gently to evenly distribute the fixation solution over the cover slip. After that, incubate the dish for 10 minutes at room temperature. Aspirate the fixation solution from the dish.

Now add two milliliters of PBS along the walls of the dish. And then aspirate the PBS. To prepare for DAPI staining, add five microliters of DAPI staining reagent onto a glass slide.

Then remove the cover slip from the dish using a scalpel. And drain excess PBS on the cover slip by touching the edge of the cover slip with a paper towel. Now mount the cover slip upside down on the drop of DAPI staining reagent on the glass slide so that the cells are exposed to the DAPI staining reagent.

Finally, examine the stained cells under a fluorescence microscope equipped with a DAPI filter. Nuclear morphologies in each of the three modes of cell death are shown. Apoptotic cells are seen having condensed nuclei.

Multiple lobes are observed in nuclei of cells undergoing mitotic catastrophe. Cells undergoing senescence are seen to display characteristic senescence-associated heterochromatin foci. A comparative observation of mock-irradiated nuclei and nuclei irradiated with six grays X-rays is shown.

Circles indicate nuclei undergoing apoptosis, and arrows indicate nuclei undergoing mitotic catastrophe. Evaluation of about 300 nuclei subjected to irradiation compared to the mock are shown. The six grays X-ray irradiation is seen to cause mitotic catastrophe more frequently at about 25%followed by apoptosis at about 5%and very rare senescence events.

Once mastered, this technique can be done in one hour if it is performed properly. While attempting this procedure, it's important to remember not to damage the cells by applying solutions directly to the cover slips. Following this procedure, other methods, such as flow cytometric analysis or cell death marker proteins, as well as senescence associated beta-galactosidase staining to assess senescence can be performed to further understand each mode of clonogenic cell death.