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

maximum of 1 h post isolation. The wells containing no compound are taken as controls.

2. Plate out a second set of primary rat hepatocytes in 200 ml growth media for 21 h prior to dosing with staurosporine.

3. Following the 21 h adherence period, dose the second set of primary rat hepatocytes with 0 µM, 0.5 µM, 1 µM, or 5 µM staurosporine respectively for 4 h in growth media. The wells containing no compound are taken as controls.

4. After the appropriate exposure period, remove the dosing media and replace it with media containing MitoTracker Deep Red 633 (25 µM) and Hoechst 33342 (1 µM) probes in a final volume of 100 µl per well for 10 min at 37 ºC, 5% CO2.

5. Image on the IN Cell Analyzer 3000 at 37 ºC using the following filter sets: Hoechst Ex. 352 nm, Em. 455 nm; MitoTracker Deep Red 633 Ex. 644 nm, Em. 665 nm.

6.Analyze the images using the granularity algorithm of the IN Cell Analyzer 3000 in both the nuclear and cytoplasmic compartments.

The protocol provided allows nuclear changes that occur during apoptosis to be tracked using the DNA binding dye Hoechst 33342. Non-apoptotic cells exhibit homogeneous nuclear staining (Fig 1A). As cytotoxicity occurs with increasing dose of compound, the staining reveals an increasingly textured punctate pattern (Fig 1B) attributable to nuclear DNA condensation. Mitochondrial changes are monitored using MitoTtracker Deep Red 633. The observed morphology changes from an intense punctate distribution of the dye (Fig 1D) to a dimmer, more diffuse pattern (Fig 1E) with increasing concentration of compound .

The Granularity Analysis Module is applied sequentially to analyze the nuclear and cytoplasmic staining. This module automatically defines measurement areas of interest such as nuclei and cell bodies (Figs 1C and 1D),
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