digoxygenin vs. PI Apoptosis
darzynk@nymc.edu
Sat, 06 May 1995 16:15:33 -0500
Dr. Claude Cautin from Montreal seeks an explanation for the observed
differences between Apoptotic Index (AI) estimated by DNA strand
breaks assay vs. DNA stainability.
Each assay to identify apoptotic cells has different "time window" to
recognize paricular type of changes which characterize apoptosis. The
longer duration of the "window", the higher is AI. DNA strand breaks
appear very early during apoptosis and therefore cell labeling with
digoxygenin can be seen before DNA is degraded to such an extent that
it can be extracted (i.e. before apoptotic cells can be recognized as
having fractional DNA content). However, at late stages of apoptosis
DNA is degraded to such an extent that even cell prefixation with
formaldehyde (crosslinking) cannot retain within the cell. With loss
of DNA there are many fewer DNA strand breaks (DNA ends).
Furthermore, DNA is lost by shedding of "apoptotic bodies". Thus, the
late apoptotic cells contain a sub/G1 DNA content and much decreased
number of DNA strand breaks and may be difficult to distinguish from
late necrotic cells. Clearly, however, AI estimated by DNA strand
break vs. DNA content analysis may be different.
Another issue that affects the estimate of AI by flow cytometry,
pertains the use of detergents.
Analysis of AI by analysis of DNA content following cells'
permeabilization with detergents, rather than fixing with ethanol, is
unreliable because, when the plasma membrane is broken (by detergent)
a single apoptotic cell may release numerous nuclear fragments. Also
unreliable is AI estimate if one is using exponential rather than
linear scale of the DNA content coordinate. A minute DNA fragments,
which can be seen by such an analysis (e.g. with DNA content lesser
than 10% of total cellular DNA) may be individual chromosomes
(released from mitotic cells treated with detergent), apoptotic
bodies, debris, etc.
Zbigniew Darzynkiewicz
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
