Cell Death Screening Using DAPI Staining: A Method for Rapid Screening of IR Induced Clonogenic Cell Death in Cancerous Cell Lines

Exposure to ionizing radiations such as X-rays can cause DNA damage, eventually leading to cell death. To screen the effect of ionizing radiations on cell death profiles, begin by taking a culture dish containing adherent cancer cells placed over the surface of a coverslip.

Irradiate the culture dish with X-rays for the desired duration. This treatment causes DNA damage inside the cells. Next, aspirate the spent media from the culture dish and add a suitable fixative. This solution permeates the cells and locks the cellular components in place during subsequent staining steps.

Discard the fixative solution. Remove the coverslip containing X-ray treated cells from the culture dish. Flip the coverslip and place it onto the surface of a glass slide carrying a drop of DAPI stain over it such that the cells are in direct contact with DAPI.

The DAPI molecules enter the nuclei and bind to the A-T-rich sites of DNA. Now, visualize the cells under a fluorescence microscope. The nuclei appear blue and exhibit distinct morphologies.

The cells showing condensed and fragmented nuclei are indicative of apoptosis, programmed cell death. Others displaying multiple-lobed nuclei represent that cells are experiencing mitotic catastrophe. Those with flattened nuclei showing multiple bright spots are suggestive of cells undergoing senescence.

Take the cells from the incubator and aspirate the culture medium from the culture dish. Cut the tip of a 1,000-microliter micropipette tip about 5 millimeters from the end using scissors, and use it to add 1 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 coverslip. After that, incubate the dish for 10 minutes at room temperature.

Aspirate the fixation solution from the dish. Now, add 2 milliliters of PBS along the walls of the dish and then aspirate the PBS. To prepare for DAPI staining, add 5 microliters of DAPI staining reagent onto a glass slide. Then, remove the coverslip from the dish using a scalpel and drain excess PBS on the coverslip by touching the edge of the coverslip with a paper towel.

Now, mount the coverslip 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.