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ARMIN WOLF ANAL REAL ACAD. FARM.

After a further washing, the ABC substrate9 was added for 5-15 min.
Cells were washed and mounted with Crystal Mount. Annexin-positive
cells were detected by their brown color.

Determination of caspase-1, -3 and -6 activity

Caspase activity was determined according to the method of
Rodriguez (Rodriguez et al. 1996).

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 inhibitors10). After three thaw-freeze
cycles, the lysate was centrifuged for 20 min at 13000 g at 4 °C. The
supernatant (lysate) was removed and stored at –80 °C until the assay was
performed.

Lysates (70 µg protein) were assayed in 0.1 % CHAPS, 100 mM

Hepes, 10 % sucrose and 10 mM DTT, pH 7.5, with or without protease

inhibitors (100 µM); caspase-1 (Ac-YVAD-CHO), caspase-3 (Ac-DEVD-
CHO) or caspase-6 (Ac-VEID-CHO)11 were added in DMSO. The

reaction was started with 20 µM of the substrate for caspase-1 (Ac-

YVAD-AMC), caspase-3 (Ac-DEVD-AMC) and caspase-6 (Ac-VEID-

AMC), which were labeled with the fluorochrome 7-amino-4-methyl
coumarin (AMC)11, and the reaction was followed for 60 min.

Fluorescence was measured at excitation 360 nm and emission 460 nm in

a fluorescence plate reader.

Fluorescence intensity was calibrated with standard concentrations
of 7-amino-4-methyl coumarin (AMC). Protease activity was calculated
from the slope of the recorder trace and expressed as pmol/ mg protein/
min.

The difference between the substrate cleavage activity levels in the
presence and absence of selective inhibitors reflected the contribution of
either caspase-1, -3 or -6 enzyme activity.

Determination of mitochondrial membrane potential

Mitochondrial membrane potential was determined by the uptake
of Rhodamine 123 according to the method of Wu (Wu et al. 1990).

8