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

and then quantitates qualifying ‘granules’ of a specified dimension detected within the measurement region. For both assays, the nuclear image is used as a starting point to define the measurement region, which can be adjusted to encompass either the entire cell body or just the nuclear area. A granule size is then chosen to best represent the biology and the punctate structures are automatically quantitated in the desired channel (blue channel for nuclear measurements; red channel for mitochondrial measurements).

During development and validation of the protocol, four additional more traditional cell-based assays were run in parallel to determine the cytotoxicity of diclofenac in primary rat hepatocytes. The assay giving the lowest EC50 value with the most reproducible data, Alamar Blue, was then taken as the benchmark assay to compare the sensitivity of the nuclear and mitochondrial status assays (1). Alamar Blue is a tetrazolium salt sensitive to cellular redox levels and is widely used to assess cytotoxicity.

For all data plots shown, the results are expressed as a percentage of control values obtained from cultures receiving no test compound. For the mitochondrial and nuclear status assays, the percentage response is calculated from the population-averaged Ngrain value, Ngrains being the number of grains detected per cell or per nucleus (depending on the assay). The Granularity Analysis Module is also used to determine cell number per image.


Results
Following exposure to staurosporine or diclofenac, primary rat hepatocytes show early signs of cytotoxicity. DNA condensation is clearly observed in treated cells compared to controls (Figs 2 and 3). There is also a distinct decrease in mitochondrial punctate fluorescence following treatment (Figs 1D and 1E). This decrease reflects changes in mitochondrial permeability that occur during apoptosis and necrosis. U
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