Apoptosis is a physiological form of death that is crucial in growth, maintenance and normal development of tissues. One of the first events in apoptosis is the isotonic shrinkage of the cells, named Apoptotic Volume Decrease (AVD). The aim of the present work was to address the possible link between anoikis (apoptosis induced by detachment from the extracellular matrix) and cell volume changes that characterize the onset of apoptosis in isolated superficial rat colonocytes. Cell suspension was obtained by incubating the everted colon in 0.5% trypsin solution in Dulbecco's Modified Eagle Medium at 30 °C for about 1 h. Cell viability after preparation was tested by Trypan blue exclusion test. Since one of the earliest indications of apoptosis is the translocation of the membrane phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane, the detection of apoptosis was performed by annexin V and propidium iodide labeling and confocal microscopy; cell volume changes were monitored by light microscopy and video imaging. The results obtained in the present work demonstrated that colonocytes underwent anoikis within a few hours after isolation, as indicated by the appearance of annexin V positivity. During the first phase of apoptosis the cells showed an early normotonic shrinkage, increasing with the time. This phenomenon can be ascribed to AVD, representing one of the major hallmark of apoptosis.
ANOIKIS AND APOPTOTIC VOLUME DECREASE IN ISOLATED RAT DISTAL COLON SURFACE CELLS
GIORDANO, Maria Elena;LIONETTO, Maria Giulia;SCHETTINO, Trifone
2012-01-01
Abstract
Apoptosis is a physiological form of death that is crucial in growth, maintenance and normal development of tissues. One of the first events in apoptosis is the isotonic shrinkage of the cells, named Apoptotic Volume Decrease (AVD). The aim of the present work was to address the possible link between anoikis (apoptosis induced by detachment from the extracellular matrix) and cell volume changes that characterize the onset of apoptosis in isolated superficial rat colonocytes. Cell suspension was obtained by incubating the everted colon in 0.5% trypsin solution in Dulbecco's Modified Eagle Medium at 30 °C for about 1 h. Cell viability after preparation was tested by Trypan blue exclusion test. Since one of the earliest indications of apoptosis is the translocation of the membrane phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane, the detection of apoptosis was performed by annexin V and propidium iodide labeling and confocal microscopy; cell volume changes were monitored by light microscopy and video imaging. The results obtained in the present work demonstrated that colonocytes underwent anoikis within a few hours after isolation, as indicated by the appearance of annexin V positivity. During the first phase of apoptosis the cells showed an early normotonic shrinkage, increasing with the time. This phenomenon can be ascribed to AVD, representing one of the major hallmark of apoptosis.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
