BACKGROUND: Apoptosis is a an important process in corneal development, homeostasis, and disease. This study was performed to determine for the first time basic temporal apoptotic features of SV-40 immortalized human corneal epithelial (HCE) cells. Additionally, we introduce a sensitive analysis of confocal microscopic images to measure the kinetics of staurosporine (STS) induced phosphatidylserine (PS) membrane translocation and early nuclear morphological changes. METHODS: HCE cells were cultured in the presence of STS to induce apoptosis. Caspase-3 activity was measured with the fluorogenic substrate z-DEVD-rhodamine 110. We determined mitochondrial viability with a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate reduction assay, and chromatin degradation with a fluorometric method using 4,6-diamidino-2-phenylindole (DAPI). Membrane translocation of PS and nuclear alterations were assessed by quantitative fluorescence microscopy. Image processing routines were written in interactive data language (IDL). RESULTS: Nuclear alterations like hyperchromicity, pyknosis, and active chromatin reorganization evolved instantly after STS induction. They were followed by PS translocation, DNA fragmentation, mitochondrial breakdown, and caspase-3 activation, which were detected between approximately 90 min and 4 h. CONCLUSIONS: Morphological and texture sensitive descriptors proved to be highly susceptible for the quantification of early apoptotic nuclear characteristics in HCE cells. We propose this method to be considered for the detection of subtle nuclear reorganization in cellular studies. Copyright 2003 Wiley-Liss, Inc.
BACKGROUND: Apoptosis is a an important process in corneal development, homeostasis, and disease. This study was performed to determine for the first time basic temporal apoptotic features of SV-40 immortalized human corneal epithelial (HCE) cells. Additionally, we introduce a sensitive analysis of confocal microscopic images to measure the kinetics of staurosporine (STS) induced phosphatidylserine (PS) membrane translocation and early nuclear morphological changes. METHODS: HCE cells were cultured in the presence of STS to induce apoptosis. Caspase-3 activity was measured with the fluorogenic substrate z-DEVD-rhodamine 110. We determined mitochondrial viability with a 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate reduction assay, and chromatin degradation with a fluorometric method using 4,6-diamidino-2-phenylindole (DAPI). Membrane translocation of PS and nuclear alterations were assessed by quantitative fluorescence microscopy. Image processing routines were written in interactive data language (IDL). RESULTS: Nuclear alterations like hyperchromicity, pyknosis, and active chromatin reorganization evolved instantly after STS induction. They were followed by PS translocation, DNA fragmentation, mitochondrial breakdown, and caspase-3 activation, which were detected between approximately 90 min and 4 h. CONCLUSIONS: Morphological and texture sensitive descriptors proved to be highly susceptible for the quantification of early apoptotic nuclear characteristics in HCE cells. We propose this method to be considered for the detection of subtle nuclear reorganization in cellular studies. Copyright 2003 Wiley-Liss, Inc.
Authors: Poonam Malik; Nikolaj Zuleger; Jose I de las Heras; Natalia Saiz-Ros; Alexandr A Makarov; Vassiliki Lazou; Peter Meinke; Martin Waterfall; David A Kelly; Eric C Schirmer Journal: PLoS One Date: 2014-11-11 Impact factor: 3.240