Morphometric quantification of apoptotic stages in cell culture.
Apoptosis is an active process of self-destruction, whereby cells undergo physiological cell death. It occurs during development and regulation of tissue homeostasis or as a result of changes in environmental stimuli. Chromatin condensation and nuclear fragmentation, which are typical features of apoptotic nuclei, are usually quantified by fluorescent DNA dyes. The present study reports a reliable method to analyze morphological apoptotic stages in cultured cells, using light microscopy. We used the human neuroblastoma cell line SK-N-BE as a model to study apoptosis induced by inadequate cell-matrix interactions. Apoptosis was detected on cells cultured for different time intervals on polyHEMA, poly-L-lysine or collagen I. Quantitative morphometric and densitometric analysis after hematoxylin nuclear staining and caspase-3 immunocytochemistry, as markers of occurring apoptosis, were performed. Our method identifies different stages of caspase-3 activation and the subsequent DNA fragmentation and condensation. This experimental procedure enables us to detect slight differences in apoptosis progression by morphological analysis.
Sabbatini
Maurizio
M
Department of Medical Science, Human Anatomy Laboratory, University of Eastern Piedmont A. Avogadro, via Solaroli 17, IT-28100 Novara, Italy.
Bozzo
Chiarella
C
Castellucci
Mario
M
Cannas
Mario
M
D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D004396:Coloring Agents; D053938:DNA Fragmentation; D003720:Densitometry; D005109:Extracellular Matrix / Q000378:metabolism; D005456:Fluorescent Dyes; D006416:Hematoxylin; D006801:Humans; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D009447:Neuroblastoma / Q000473:pathology; D011107:Polylysine / Q000378:metabolism; D011419:Propidium; D013997:Time Factors
D024042:Collagen Type I; D004396:Coloring Agents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011107:Polylysine; D011419:Propidium; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D006416:Hematoxylin