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First of all, "TUNEL" name is a misnomer. "N" in TUNEL stays for DNA
nicks. "Nicks" denote breakage in a backbone of a single strand of
the DNA helix. Most DNA lesions during apoptosis are DNA strand
breaks. Double stranded breaks are clearly revealed by "laddering"
during gel electrophoresis. The method utilizing terminal
transferase, thus, labels predominantly DNA breaks and not nicks.
Actually, the "nick translation" methodology, utilizing DNA
polymerases rather than terminal transferase would be more
appriopriate to label the "nicks".
Regarding cells' loss. Prefixation in ethanol followed by
formaldelyde may contribute to such a loss. We fix the cells in 1%
formaldehyde (15 min), then rinse and postfix (store) in 70% ethanol
(Gorczyca et al., Int. J. Oncol., 1:639, 1992; Leukemia, 7:659
1993). After fixation, all centrifugations and incubations with TdT
are done in the same tube (in which the cells were initially fixed;
transfering cell suspensions to new tubes causes additional cell
loss). Using increased concentration of serum albumin during washes
and centrifugations decreases somewhat cell loss. In critical
situations, when one has very few precious cells to begin, I would
suggest to use carrier cells (e.g. dilute the cells with chick or
fish erythrocytes), the later can be identified during analysis based
on differences in DNA content (we did not test this step, however).
As I have mentioned already at this forum, the single step DNA strand
breaks labeling procedure has fewer centrifugations, and thus causes
lesser cell loss (will be published in June issue of Cytometry).
Good luck in labeling DNA strand breaks (and if one wishes, the nicks
too).
Zbigniew Darzynkiewicz
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