Resources Technical Documents live cell imaging of apoptosis in ECM-detached epithelial cells in CyGEL™
Technical documents description.....
Background: Apoptosis is a stochastic process and therefore requires long periods of imaging to allow capture of this short-lived event. Anoikis is a model of apoptosis where cells detached from the extra-cellular matrix undergo the events associated with apoptosis but in a reversible way. Anoikis can be observed in epithelial cells. The aim of the research was to better understand the control and regulation of apoptosis involving the Bcl-2 protein Bax. One aspect of this was to show whether the kinetics of apoptosis (as determined by time for translocation of a YFP-SMAC signal from cytosol to mitochondria) was dependent upon the time elapsed following detachment and before apoptosis began. These constraints would necessitate imaging live cells for up to several hours without allowing cell-cell or cell-surface contact (and any resulting stimuli), keeping such non-adhered cells immobilised yet otherwise viable to permit images to be captured every 3 minutes until apoptosis and observation of the symptomatic membrane blebbing and nuclear condensation. Additionally, since the epithelial cells were highly dependent on suitable medium (DMEM F12) and growth factors (EGF, insulin) this environment had to be provided throughout the imaging period. Accordingly, CyGEL™ was utilised as live cell mountant compatible with the requirements of the experimental procedure.
Results: As shown in the paper, viable cells were imaged for several hours without necrosis. Cells, however, underwent the expected series of events in apoptosis following the translocation of (YFC-)SMAC. The kinetics of this translocation were found to be independent of the time from detachment and were in line with previous data on apoptosis initiated by staurosporine or UV irradiation.
Conclusions: CyGEL™ was been demonstrated as an immobilising support compatible with viable cells and fluorescence-based imaging of such cells over several hours. This immobilisation kept cells still and free from the interference of cell-cell interaction and contact with the surface of the imaging chamber. CyGEL™ was successfully supplemented with media and growth factors critical for normal cell survival, permitting the observation of stochastic apoptosis-priming events.
Reference: Upton J-P, Valentijn AJ, Zhang L, Gilmore AP. The N-terminal conformation of Bax regulates cell commitment to apoptosis. Cell Death and Differentiation (2007) 14, 932–942. Wellcome Trust for Centre for Cell Matrix Research, Faculty of Life Sciences, The University of Manchester, UK.
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