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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Assess the In Vivo Activation of Signal Transduction Pathways with
PathDetect Reporting Systems2.
Ca++-Induced G Protein Coupled Receptor (GPCR) Activation Monitored via Aequorin Luminescence in the LMax Microplate Luminometer (MaxLine Application Note #42)3.
FLIPR Calcium 3 Assay Kit - An Optimal No-Wash Fluorescence Calcium Indicator Reagent for Non-Optimal Calcium Responses4.
Monitoring Receptor-Mediated Changes in [Ca2+]i Using Single-Wavelength and Dual-Wavelength Indicators on the FlexStation Workstation5.
RT-PCR Primers for the Study of Apoptosis6.
A Further Step in Understanding Apoptosis
Direct Detection of PARP Cleavage7.
Apoptosis detection by annexin V and
active caspase-3 with the Agilent 2100
bioanalyzer8.
Promegas Multiplexed Cell Viability and Apoptosis Assays performed on the PHERAstar9.
An Image-Based Assay of Endothelial Cell Tube Formation as a Model of Angiogenesis10.
Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations11.
Matched siRNAs and Assays: Ambion + Applied Biosystems = RNAi Success