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VOL 66 (4) 2000 IN VITRO INDUCTION OF APOPTOSIS BY CYCLOSPORINE A

After treatment, hepatocytes cultured on 96-well plates1, were
washed in PBS and incubated with 10 Pg/ml Rhodamine 12312 for 30 min
at 37 °C. After further washing, the hepatocytes were incubated with
WME medium for 30 min. Ethanol/Water 1/1 was used to extract the
amount of dye retained by the cells. Fluorescence was measured with a
Cytofluor 2300 from Millipore, with an excitation wavelength of 485 nm
and an emission wavelength of 530 nm.

Determination of cytosolic and mitochondrial cytochrome c content

Cytochrome c was determined after subcellular fractionation by
the Western blotting technique according to the method of Tang (Tang et
al. 1998).

After incubation, 2 u 106 cells were washed once in ice-cold PBS
and lysed in 1 ml buffer A (10 mM Hepes, pH 7.4, 42 mM KCl, 5 mM
MgCl2, 1 mM DTT and protease inhibitor. After three thaw-freeze cycles,
the lysate was centrifuged for 20 min at 13 000 g at 4 °C. The pellet
fraction (mitochondria) was first washed in buffer A containing sucrose
and then solubilized in 50 µl TNC buffer (10 mM Tris-acetate, pH 8.0,
0.5 % NP-50, 5 mM CaCl2) The supernatant was re-centrifuged at 100
000 g (4 °C, 1 hour) to generate the cytosol, and then removed and stored
at -80 °C.

Fifty µg of the cytosolic and mitochondrial fraction was loaded
onto a 15 % SDS- polyacrylamide gel and separated according to
Laemmli (Laemmli 1970). After fractionation, the proteins were
electroblotted onto a PVDF transfer membrane (0.2 µm)13.
Immunostaining of cytochrome c was carried out with an anti-cytochrome
c monoclonal antibody14. For detection, the membrane was washed in
PBS and incubated with Streptavidin peroxidase complex for 30 min.
After further washing, the ABC substrate was added for 5-15 min. Protein
bands (15 kD) were revealed using NBT/BCIP.

Determination of protein concentration

Protein content was determined according to Bradford (Bradford
1976). Bovine serum albumin served as standard.

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