This article details a protocol for inducing apoptosis in Jurkat T lymphocyte leukemia cells using ultraviolet C (UVC) radiation. The process involves assessing cell viability, exposing cells to UVC, and analyzing apoptotic markers through flow cytometry.
Take a suspension of Jurkat cells — T lymphocyte leukemia cells.
Mix an aliquot with a cell viability dye and ensure an adequate number of living cells.
Plate the cells in a culture dish and apply ultraviolet C or UVC, radiation for the required duration.
UVC penetrates the cells and is absorbed by DNA, forming pyrimidine photoproducts.
Post-irradiation, incubate the cells. Extensive UVC-mediated DNA damage initiates the intrinsic apoptosis pathway, activating pro-apoptotic proteins on the outer mitochondrial membrane.
Activated proteins oligomerize, forming membrane pores and releasing cytochrome C from the intermembrane space into the cytoplasm.
Cytochrome C complexes with the apoptotic protease activating factor-1, forming an apoptosome complex. The apoptosome activates caspases, causing apoptosis.
Introduce fluorophore-labeled annexin V to label phosphatidylserine on the membrane, an apoptotic cell marker, and add propidium iodide that enters through the damaged membrane to label the DNA.
Using flow cytometry, determine the annexin V-labeled early apoptotic cells and dual-stained late apoptotic cells.
Begin by culturing Jurkat T cells in basal cell culture medium, supplemented according to the manuscript directions, at 37 degrees Celsius and 5% carbon dioxide. Jurkat T cells are a suspension cell line that can be maintained through passaging into pre-warmed culturing media every three days.
Prepare apoptotic cells by growing them to 90% confluency, which takes three to four days after passaging. Grow cells in five T75 flasks, to obtain a sufficient number of cells for this protocol. Once the cells are ready, aspirate the flask contents about 24 milliliters, and transfer them to a 50-milliliter conical tube. Count the cells using trypan blue staining and a hemocytometer.
Pellet the cells by centrifugation at 271 times g for five minutes, and discard the supernatant. Then, resuspend the cells in media to obtain three million cells per milliliter. Aliquot the cells into 100-by-20 millimeter tissue culture dishes by transferring five milliliters of cells per dish.
Prepare approximately nine culture dishes. Set one dish aside as an unexposed control, and expose the remaining dishes to UV. Set the UV crosslinker to the correct energy level by pressing the energy button and entering "600" with the number pad. Irradiate all dishes with the cells except for the control, making sure to remove the top cover of the tissue culture dish during UV exposure.
Then, incubate all the dishes in a cell culture incubator at 37 degrees Celsius and 5% carbon dioxide for four hours. After the incubation, combine all the irradiated cells into a 50-milliliter conical tube, and pellet them by centrifugation at 271 times g for five minutes. Discard the supernatant, and resuspend the cells in 24 milliliters of sterile PBS. Centrifuge the tube again, and remove the supernatant.
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