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

distilled water, and mounted by Crystal Mount5. After staining,
hepatocyte nuclei were violet in color.

The following criteria were used: normal nuclei were those in
which the chromatin was unaltered and uniformly spread over the whole
nucleus. Condensed chromatin was located at the nuclear membrane
periphery and appeared in a half-moon form. Fragmented chromatin was
identifiable by its scattered, droplike structure, which was located on the
area of the original nucleus. The total size of apoptotic nuclei appeared to
be smaller and more shrunken when compared with intact cells. For each
sample, 1000-1500 nuclei were counted.

Determination of DNA fragmentation

TUNEL assay was performed using the DNA fragmentation kit
TdT-FragELTM 6. The principle of the assay is based on the fact that
terminal deoxynucleotidyl transferase (TdT) binds to exposed 3’-OH ends
of DNA fragments generated in response to apoptotic signals, and
catalyzes the addition of biotin-labeled and unlabeled deoxynucleotides
(Gavrieli et al. 1992). Biotinylated nucleotides were detected using a
streptavidin-horseradish peroxidase (HRP) conjugate. Diaminobenzidine
reacts with the labeled sample to generate an insoluble, colored substrate
at the site of DNA fragmentation. Non-apoptotic cells do not incorporate
significant amounts of labeled nucleotide because they lack an excess of
3’-OH ends. Non-apoptotic cells were counterstained with methyl green.
Positive TUNEL staining was indicated by a dark brown DAB signal,
while shades of blue-green signified a non reactive cell.

Determination of membrane phosphatidylserine distribution

Phosphatidylserine distribution was detected by labeling the cells
with the biotin-conjugate of Annexin V7 according to the method of
Vermes (Vermes et al. 1995).

Cells were washed with binding buffer (Hepes/NaOH 10 mM pH
7.4, NaCl 140 mM, CaCl2 2.5 mM) and incubated for 1 hour with
Annexin-biotin 1:20 in binding buffer. After washing in PBS, the cells
were fixed with formalin. To detect of Annexin V, cells were washed in
PBS and incubated with Streptavidin peroxidase complex8 for 30 min.

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