Hi James, I always tell my users that we have to be careful about any of the methods used for looking for apoptosis by flow when we say that they are 'specific'. We have to remember that we are looking for signs that apoptosis may have occurred by looking for 'symptoms' of it. In the sub-G1 method, a peak to the left of the G1 peak will be found if there are cells that have fragmented their DNA into small enough fragments to have these eluted by the washing steps. This is generally a late stage event and may also occur in necrotic cells which will be indistinguishable from the ones that are apoptotic. The advantage of the sub-G1 method is that it is quick, cheap and easy and can give a good idea that something is happening, which can then lead on to using other methods such as TUNEL that are more quantitative (in that it quantitates strand breaks not necessarily apoptotic cells). It is probable that the figures found for % apoptotic cells will differ with each method used as each is measuring a different point in the apoptotic pathway and some methods will also be including necrotic/late stage apoptotic cells as well. In all cases it is de rigeur to correlate flow cytometric findings with morphology. Derek On Tue, 17 Aug 1999, James Liou wrote: > To those with expertise and experience using PI to look at sub 2N DNA > content as a measurement of apoptosis, I have a fairly basic question that > I hope you can answer. Does this assay definitively distinguish between > apoptosis and necrosis? > I guess these days suspicion arises in the field of cell death because > almost everything is decribed as apoptosis. I just don't know if the PI > hypodiploid assay gives one the same DNA fragmentation readout as say TUNEL > or a laddering assay. If the PI assay is specific for apoptosis, what > would a necrosis profile look like in comparison? > Thanks in advance to any who can help me with an answer. This group is > truly fantastic! ************************************************************************ Derek Davies Voice: (44) 0171 269 3394 FACS Laboratory, FAX: (44) 0171 269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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