Quantification of apoptosis by propidium iodide incorporation and caspase 3 activity.

<p><i>A</i>. In the MEKK1^mutP and MEKK1^mutK cell lines the percentage of apoptotic cells does not significantly increase after cytoskeletal disruption. To quantify the levels of DNA fragmentation, propidium iodide staining was analyzed by flow cytometry. Cell lines were treated with 1 µM vinblastine (panel I), 2 µg/ml cytochalasin D (panel II) or 90 nM okadaic acid (panel III) for twelve hours and the percentage of apoptotic cells were determined by measuring fluorescence lower than the diploid population using CellQuest software. <i>B</i>. Caspase 3 is not activated in the MEKK1^mutP or MEKK1^mutK cell lines after cytoskeletal disruption. Caspase 3 activity was determined after four hours of treatment with 1 µM vinblastine (I), 2 µg/ml cytochalasin D (II) or 90 nM okadaic acid (III) treatment. Cell lysates were used to determine the catalytic activity of caspase 3 by using the colorimetric substrate Ac-DEVD-pNA and measured at 405 nM.