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

sing the IN Cell Analyzer 3000 system and software it is possible to detect and quantitate these changes.

Staurosporine treatment
Cells exposed to staurosporine for 4 h are viable at all exposure concentrations as shown by the Alamar Blue assay data (Fig 4). Even at the highest dose, there is no significant change in Alamar Blue signal relative to controls. The cell number also remains fairly constant, dropping to 76% of controls only at the highest dose of staurosporine. However, the nuclear measurements show concentration-dependent changes in nuclear texture consistent with DNA condensation (Fig 4). Dose-dependent decreases in mitochondrial staining with MitoTracker Deep Red 633 are also observed. Thus, although little or no toxicity is detected using traditional measurements such as cell number and Alamar Blue, the nuclear and mitochondrial measurements obtained using IN Cell Analyzer 3000 clearly indicate that staurosporine-induced cell death is well underway.

Diclofenac treatment
Similar to staurosporine treatment, diclofenac treatment results in little change in the Alamar Blue data; cells are viable at all concentrations of diclofenac after 21 h of exposure (Fig 5). Cell numbers do not deviate statistically from control values at any of the compound concentrations. DNA condensation; however, increases at the highest dose of diclofenac to 240 % of control values, while punctate mitochondrial staining decreases at the highest dose of diclofenac to less than 60% of control values. Indications from the Alamar Blue reference assay and cell number measurements are that there is no overt toxicity occurring in the system at any dose of diclofenac. By contrast, it is clear from the data acquired with the IN Cell Analyzer 3000 system that cytotoxicity can be detected at 300 mM diclofenac.

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