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Caspase-3 Activation - An Indicator of Apoptosis in Image-Based Assays

d in cellular assays to quantify activators and inhibitors of the death cascade. The response is both time and concentration dependent suggesting that multiple pathways play a role in triggering the caspase-3 activation. One could hypothesize that cells are most susceptible to staurosporine in a specific phase of the cell cycle and therefore over time, most cells will die, similar to the findings of Vermeulen et. al. (2003).

We show that this representative apoptosis assay can be used in an automated way using the BD Pathway Bioimager and its built-in image and data analysis tools making it amenable to a high-throughput application development. Our data supports findings by Jessel et. al. (2002) who report an EC50 for staurosporine of 200 nM. We specifically see the usefulness of this assay as a building block in a multiplexed high-content assay. This caspase-3 assay can easily be accommodated in a different fluorescence channel and since the BD Pathway Bioimager utilizes broad-spectrum lamps, many dye combinations are possible. The BD Pathway Bioimager and its software tools provide the optimal environment to conduct this kind of multidimensional analysis.

References

1. Jacobson, M.D., Weil, M., Raff, M.C.. 1996. Role of Ced-3/ICE-Family Proteases in Staurospirine-induced Programmed Cell Death. JCB , 133:1041-1051.

2. Lavrik I.N., Golks, A., Krammer, P.H.. 2005. Caspases: pharmacological manipulation of cell death. J. Clin. Invest. 115:10. 2665-72.

3. Nicholson D.W. Ali A., Thornberry N.A. et al . 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37-43.

4. Vermeulen, K., Berneman, Z.N., Van Bocksaele, D.R. 2003. Cell cycle and apoptosis. Cell Proliferation, 36:165-175.

5. Jessel, R., Haertel, S., Socaciu, C. Tykhonova, S., Diehl, H.A.. (2002). Kinetics of apoptotic markers in e
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