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The measurement of DNA condensation and mitochondrial changes during apoptosis and necrosis in cell based assays

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Conclusions
The IN Cell Analyzer 3000 system and software can be used successfully to quantitate quantify changes in nuclear and mitochondrial status following treatment with apoptosis-inducing agents such as staurosporine and diclofenac. Assays assessing nuclear and mitochondrial status can be multiplexed, thereby reducing the quantities of primary cells and compounds required. Rapid, sequential data analysis allows the results for each of the multiplexed assays to be generated in approximately 0.2 s per image, representing a significant time-savings over more traditional methods, and thereby enabling high-throughput assessment of cytotoxicity.

Nuclear DNA condensation, typical of late stage apoptosis, and mitochondrial changes are clearly visible and can be quantitated at the lowest concentration of staurosporine (0.5 µM) and at 300 µM diclofenac. By contrast, the Alamar Blue assay is not capable of detecting cytotoxic effects relative to controls even at the highest concentrations of these compounds. Thus, DNA condensation and mitochondrial changes are more sensitive indicators of cytotoxicity than the conventional Alamar Blue assay.

Multiplexing DNA condensation and mitochondrial status may be useful in determining mechanisms of drug action. In addition, the multiplex format has the advantage that each assay is performed on the same population of cells, thereby increasing confidence in the correlation between endpoints.


References
1. Feng, G. and Kaplowitz, N. Mechanism of staurosporine-induced apoptosis in murine hepatocytes. Am J Physiol Gastrointest Liver Physiol. 282(5), G825–34 (2002).

2. Sanchez, V. et al. Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes. Biosci Rep. 12(3), 199
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