TUNEL assay - summary of causes of variability

From: Shari Brezinsky (Shari_Brezinsky@biogen.com)
Date: Tue Jun 04 2002 - 14:33:27 EST


Dear Flowers,

I thought that I had included a summary in my last note, but apparently
my pasting was not as good as my cutting.  So here is my second try:

Shari
Biogen

Steve Mullen<sfm5ff@missouri.edu>:
I have used the "Death Detection Kit" from Roche with consistent results.
The instructions are straightforward and simple.

Dennis J. Young <djyoung@ucsd.ed>:
We've see the same thing. There is enzymatic amplification of signal, so
unless you have VERY good techniques, you will see great variation.

Zbigniew Darzynkiewicz <DARZYNK@nymc.edu>:
Apoptotic cells are strongly labeled in TUNEL assay only when they have a
large number of DNA strand breaks. This is the case when DNA fragmentation
is very extensive, which occurs when internucleosomal DNA sections are
cleaved.  However, in some cell types (often of epithelial or fibroblast
lineage) or instances  DNA fragmentation stops at the initial step i.e.
generating 300 - 50 kb DNA sections, and does not progress into
intenucleosomal (~180 bp) sections. In the TUNEL assay such cells are only
weakly labeled.

It should be noted that with most TUNEL kits (including APO-BRDU) a
positive
control cells are provided. They are camptothecin-treated leukemic cells
which are expected to have internucleosomal DNA cleavage in about 30-40 %
of
the cell population (S phase). This control, if found to be TUNEL
positive,
provides an assurance that the kit is OK. Furthermore, in the case when
the
control is positive but the investigated apoptotic cells are negative, it
provides evidence that DNA fragmentation in the studied cells did not
progress into internucleosomal DNA sections.

There are often  trivial, technical reasons for irreproducibility.
One relates to the fact that some investigators collect only adherent
cells
that are harvested by trypsinization or EDTA, discarding the medium from
flasks. Because most apoptotic cells detach, they are discarded with the
medium. The "efficiency" of discarding medium may vary from flask to
flask,
hence the variability in frequency of apoptotic cells.The correct way, of
course, is to pool the cells from medium with the these harvested by
trypsinization.

Apoptotic cells are very fragile and often completely disintegrate during
centrifugations. Hence, their frequency (apoptotic index; AI) may vary
depending on number of cell rinses, centrifugal force and time, etc. Also,
compared to nonapoptotic cells, apoptotic cells have different propensity
to
attach to the tubing. The variability in handling the samples (including
the
absolute number of total cells per sample) is then reflected by
differences
in AI.

Apoptotic cells are strongly labeled in TUNEL assay only when they have a
large number of DNA strand breaks. This is the case when DNA fragmentation
is very extensive, which occurs when internucleosomal DNA sections are
cleaved.  However, in some cell types (often of epithelial or fibroblast
lineage) or instances  DNA fragmentation stops at the initial step i.e.
generating 300 - 50 kb DNA sections, and does not progress into
intenucleosomal (~180 bp) sections. In the TUNEL assay such cells are only
weakly labeled.
It should be noted that with most TUNEL kits (including APO-BRDU) a
positive
control cells are provided. They are camptothecin-treated leukemic cells
which are expected to have internucleosomal DNA cleavage in about 30-40 %
of
the cell population (S phase). This control, if found to be TUNEL
positive,
provides an assurance that the kit is OK. Furthermore, in the case when
the
control is positive but the investigated apoptotic cells are negative, it
provides evidence that DNA fragmentation in the studied cells did not
progress into internucleosomal DNA sections.



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