Assessment of secondary necrosis of Jurkat cells using a new microscopic system and double staining method with annexin V and propidium iodide.

Using a new system developed by us for acquiring microscopic images automatically, we compared the morphological changes that apoptotic cells undergo with changes in the staining pattern of annexin V-enhanced green fluorescent protein (AV-EGFP) and propidium iodide (PI) in individual cells. Jurkat cells were treated with 5 mM CaCl2 alone, anti-Fas antibody and heating at 42 degrees C for 30 min or 46 degrees C for 60 min, and then were incubated in medium with 5 mM CaCl2. Time-lapse DNA fragmentation analysis and morphological observation revealed that the anti-Fas antibody and heating at 42 degrees C for 30 min induced typical apoptosis in the cells, and heating at 46 degrees C for 60 min induced typical necrosis. Time-lapse observation of individual cells stained with AV-EGFP and PI confirmed that apoptotic cells were stained at first with AV-EGFP alone, and thereafter also with PI when the cellular membrane ruptured and the cell underwent secondary necrosis. Most of the cells which underwent necrosis were stained simultaneously with AV-EGFP and PI. There was a significant time interval between the staining of individual cells with AV-EGFP, indicating apoptosis, and staining of these cells with PI, which indicated the occurrence of secondary necrosis. These results suggest that time-lapse examinations are necessary to distinguish apoptosis, secondary necrosis and necrosis in cells from one another. This study presents direct evidence that apoptotic cells undergo secondary necrosis, which could be recognized with PI.