Re: TUNEL vs Annexin V

Hi Andy,

I think it may be a little harsh to criticise the annexin technique 
although suggestions for other apoptosis analytical techniques might 
be welcome. What we have to remember in the detection of apoptosis by 
flow is that in general we are using a method that will detect some 
part of the apoptotic cascade, whether that be PS flipping by 
annexin, changes in mitochondrial membrane potential, cytochrome c 
release, caspase activation or changes in DNA (fragmentation etc). 
The method that you use will depend on lots of variables - cell type, 
method of apoptosis induction, facilities available and expertise 
available to name a few. One advantage of the annexin method is that, 
as it is a live cell method, you have the ability to distinguish 
between early apoptosis (annexin +ve, PI -ve) and late apoptosis 
(Annexin +ve, PI +ve) and, as you say, using time as a variable as 
well can give you more information.

TUNEL staining looks for strand breaks in the DNA which may be a late 
apoptotic event (unless apoptosis is induced by DNA damage in which 
case they will be early but contra-indicated as a marker of 
apoptosis!). One advantage is that if you combine it with a DNA dye 
such as DAPI you can get an idea as to which cell cycle phase the 
strand breaks appear. As the cells are PFA fixed, you do lose the 
ability to positively exclude dead cells from the assay. The assay is 
technically more demanding (and more expensive) so I tend to suggest 
it only if the researcher is specifically looking for strand breaks 
or has a need to see if they are cell cycle dependent. A lot of 
reviewers will ask for more than one method for assesment of 
apoptosis but they don't all have to be flow-based! The 'sub-G1' 
method will also give an idea of DNA fragmentation and although the 
information wont be as clear-cut as TUNEL in a lot of instances, it 
is very quick and easy.

Hope that helps - good luck!
Derek


>Dear flowers
>We normally use PI and Annexin V for our apoptosis studies and the 
>results are fine, with a clear distinction between early and late 
>apoptosis. This is also combined with analysis over a time course.
>For a recent paper a reviewer criticised the technique and suggested 
>using the TUNEL assay with DAPI.
>What is the advantage of this assay and what does it show that is 
>more interesting ?
>Any thoughts would be appreciated.
>
>Andy
>--


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Derek Davies					Voice: (44) 020 7269 3394
FACS Laboratory,			FAX: (44) 020 7269 3100
London Research Institute,		e_mail: derek.davies@cancer.org.uk
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