The TUNEL technique will detect nicks in DNA associated with DNA cleavage into the 300-kbp and 55-kbp fragments as well as in any subsequent DNA fragmentation down to the 182-bp multimers. However, not all DNA strand breaks are associated with apoptosis. Nicks can be seen in necrotic cells and can also be induced mechanically during sample preparation. The specificity of PS detection for apoptosis is reliant on apoptotic cells retaining plasma membrane integrity. During necrosis, the cell membrane is ruptured, allowing annexin V to enter the cell and bind to PS on the inner leaflet of the plasma cell membrane. Hence, necrotic cells appear positive for PS. This problem can be overcome by counterstaining cells with PI, which is excluded from apoptotic cells. Therefore, necrotic cells are positive for PI and annexin V; apoptotic cells are annexin V-positive but exclude PI. However, normal B cells can also be positive for annexin V (8).
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