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

damage and covalent protein-adduct formation. A toxic metabolite formed from diclofenac by the action of the cytochrome P450 system has been reported to induce apoptosis with nuclear condensation (3).


Materials
Products used
IN Cell Analyzer 3000 25-8010-11

Granularity Analysis Module
(for IN Cell Analyzer 3000) 63-0048-97


Other materials required

Primary rat liver cells (Bowman Research UK)

96-well, black, flat-bottom microtiter plates (Packard View Plate, Packard).

Hoechst 33342 (Molecular Probes)

MitoTracker™ Deep Red 633 (Molecular Probes)

Staurosporine (Sigma)

Diclofenac (Sigma)

Growth media:
1:1 mix of Williams’ E and Hams F-12 with additions of 5 µg/ml Insulin,
5 µg/ml Transferrin, 5 µg /ml Selenium, 10 nM Dexamethasone, 2 % v/v fetal calf serum,
10 mM HEPES, 50 U/ml Penicillin G, 50 µg/mlStreptomycin sulfate.

All tissue culture reagents were obtained from Gibco BRL, UK unless otherwise stated.

Fetal bovine serum (Sigma)

Phosphate buffered saline (PBS) solution

Alamar Blue™ (Serotec)


Methods
1. Plate out one set of freshly isolated primary rat hepatocytes in growth media containing 0 µM, 50 µM, 150 µM, or 300 µM diclofenac in a total volume of 200 ml in a 96-well plate for 21 h. The seeding density is 20 000 cells per well. Cells are acceptable at a minimum of 80% viability immediately prior to culture at a
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