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An In Vitro Technique for UV Irradiation-Mediated Generation of Apoptotic T Cells


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.

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