( damage and covalent protein-adduct form...)The measurement of DNA condensation and mitochondrial changes during apoptosis and necrosis in cell based assays

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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