Encyclopedia of Experiments: Immunology
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Transcript
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.
