( Key words: In Ce...)The measurement of DNA condensation and mitochondrial changes during apoptosis and necrosis in cell based assays

Key words: In Cell Analyzer • apoptosis • multiplexing • cell based assays

Apoptosis represents a gene-directed, morphologically and biochemically distinct form of programmed cell death. Inappropriate control of apoptosis is associated with a variety of disease states such as cancer, AIDS, neurodegenerative diseases, and ischaemic stroke. Hence, the mechanism and control of apoptosis is extremely important when considering the effects of new drugs on the body, or when targeting the process itself for therapeutic purposes.

The IN Cell Analyzer 3000, a high-throughput confocal imaging system, provides a means of real time, nondestructive quantitation of morphological and biochemical cellular changes. Several different endpoints can be monitored simultaneously on a cell-by-cell basis or by using a population average. Compared to more traditional single-plex techniques, multiplexed data acquisition increases the reliability of the information because data from multiple assays are derived from the same population of cells. This has the added benefit of making the most of scarce tissue/cell samples. Samples can be analyzed in a real-time or off-line mode using robust and flexible analysis modules.

In this application note, two assays are multiplexed to monitor cytotoxicity in primary rat hepatocytes. After exposure to known inducers of apoptosis, IN Cell Analyzer 3000 is used to acquire images and quantitate changes in nuclear and mitochondrial status. Primary rat hepatocytes are exposed to staurosporine or diclofenac for 4- and 21-h respectively. Staurosporine is a microbial alkaloid, a strong inhibitor of protein kinases, thought to work through JNK1, caspase proteases, AP-1, and NFκβ. It is an apoptotic inducer causing nuclear condensation (1, 2). Diclofenac is a non-steroidal anti-inflammatory compound that has been associated with liver
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