Apoptosis, also known as programmed cell death, is one of the built-in defense mechanisms of cells and plays an important role in the normal development of organisms and resistance to disease. Abnormal apoptotic processes and pathways can cause a variety of diseases, including tumors. At present, there are at least a dozen methods for detecting apoptosis. From a large framework, they can be divided into three categories based on cell morphology, biological functions, and biochemical markers. The Annexin V / PI double staining method is the best for plasma membrane analysis by flow cytometry.
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Phosphatidylserine (PS) is normally located inside the cell membrane, but in the early stage of apoptosis, PS can flip from the inside of the cell membrane to the surface of the cell membrane and be exposed to the extracellular environment. Phosphatidylserine translocation occurs in the early stages of apoptosis, prior to changes in the nucleus, DNA breakage, and cell membrane blistering. Phagocytic cells in the body can clear apoptotic cells by recognizing phosphatidylserine. Annexin-V is a Ca2 + -dependent phospholipid binding protein with a molecular weight of 35 ~ 36KD. It can specifically bind to PS with high affinity. When cells are dying or necrotic, Annexin-V can be positive (early necrotic cells may be negative) , Is a sensitive indicator for detecting early cell apoptosis. Annexin-V is labeled with fluorescein (FITC, PE), and the occurrence of apoptosis can be detected by flow cytometry or fluorescence microscopy. Propidine iodide (PI) is a kind of nucleic acid dye, it can not penetrate the intact cell membrane, but in the middle and late apoptotic cells and dead cells, PI can penetrate the cell membrane to stain the nucleus red. Therefore, matching Annexin-V with PI can distinguish early and late apoptotic cells and dead cells.
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