TUNEL-reply
darzynk@nymc.edu
Tue, 18 Jul 1995 13:15:43 -0500
Dr. K. Ton asks what may be the reason that he cannot detect
apoptosis (DNA strand breaks) in liver cells using TUNEL assay.
He mentions that he fixes cells in 2% formaldehyde (no time of
fixation is given).
Fixation step is critical. We have found that 15 min fixation in 1%
methanol-free formaldehyde on ice, followed by cell rinse in PBS and
postfixation in cold ethanol are optimal (Gorczyca et al., Int. J.
Oncol., 1: 639-648, 1992). Too high concentration of formaldehyde
and/or longer fixation times perhaps crosslink DNA too extensively
which results that its accessibility to TdT and substrates may be
decreased. Strand break labeling is then less efficient.
By the way, "TUNEL" is a misnomer - it does not provide accurate
description of the apoptotic DNA lesion which is being detected in
the assay utilizing TdT. "N" in TUNEL stays for "nicks". DNA lesions
in apoptotic cells detected by TdT, however, are double strand breaks
rather than nicks ("nick" denotes a break in the single strand of the
double stranded helical DNA). To be precize, thus, TUNEL, should be
"TUBEL", or rather TUBL.
Zbigniew Darzynkiewicz
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