A54
A real-time imaging analysis of cell death observing caspase activity and propidum iodide accumulation in live cells
David Jamieson, Jane L Armstrong, Sophie Weatherhead, Trevor R Jackson
Newcastle University, Newcastle upon Tyne, UK
Background
The detection of apoptotic death as a biological output is a central aspect of preclinical cancer research, used both in the search for novel chemotherapeutic agents, and the identification of new molecular targets. However, established endpoint assays are poorly suited to investigating the dynamic processes that underlie cell death. We describe a time-lapse microscopy imaging assay whereby both caspase activity and propidium iodide accumulation in individual cells can be observed over time and quantified.
Method
SH-SY5Y cells were plated in optical 96 well plates and incubated with 13 cis RA to promote differentiation and fenretinide to promote apoptosis. Hoescht, Nucview488 and propidium iodide were added to all wells. Nucview488 is a cell permeable caspsae 3 substrate linked to a DNA dye. Caspase 3 activity cleaves the substrate releasing the DNA dye, which translocates to the nucleus and stains DNA. The cells were imaged every hour on a BD Pathway HT automated microscope at 37oC and 5% CO2 for 12 hours. All treatments and controls were imaged in parallel.
Results
Both caspase activity and PI positive nuclei accumulated over time after addition of fenretinide to 32% and 24%, respectively, of undifferentiated cells at 12 hours. In differentiated cell populations treated with fenretinide and undifferentiated populations not treated with fenretinide less than 5% of cells were apoptotic at 12 hours. Propidium iodide accumulation lagged behind detectable caspase activity in all cells.
Conclusion
The method described allows the visualisation and quantification of apoptosis on a cell by cell basis over time.
