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 automat ically, we compared the morphological changes that apoptotic cells undergo with changes in the staining pattern of annexin V-enhanced green fluorescen t protein (AV-EGFP) and propidium iodide (PI) in individual cells. Jurkat c ells were treated with 5 mM CaCl2 alone, anti-Fas antibody and heating at 4 2 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 morpho logical observation revealed that the anti-Pas antibody and heating at 42 d egrees 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 cell s were stained at first with AV-EGFP alone, and thereafter also with PI whe n the cellular membrane ruptured and the cell underwent secondary necrosis. Most of the cells which underwent necrosis were stained simultaneously wit h AV-EGFP and PI. There was a significant time interval between the stainin g of individual cells with AV-EGFP, indicating apoptosis, and staining of t hese cells with PI, which indicated the occurrence of secondary necrosis. T hese results suggest that time-lapse examinations are necessary to distingu ish apoptosis, secondary necrosis and necrosis in cells from one another. T his study presents direct evidence that apoptotic cells undergo secondary n ecrosis, which could be recognized with PI.