Nick, I agree with what you say. In the <mumble> years that I have been doing DNA analysis by flow I remain still not entirely convinced by "doublet discrimination" - quite often whether one uses peak v integral or width v integral there is a continuum between where the G2 cells and the doublets are expected. I would agree that the smaller the beam the better you can distinguish between single cells, doublets, aggregates etc. (Whatever happened to slit scanning?). The discrimination is also affected by whether you are dealing with nuclei or whole cells and the cell type under investigation. Without modification to commercial machines, which is what the majority of investigators will have access to, what can be done? I am wary of putting exact percentages on the G2 populations (or more accurately considering that that percentage is G2 cells only), although, of course, that is what I am requested to do! It has been suggested that using a mathematical model that accounts for >G2 aggregates as well as polyploidy is a better way forward (ModFit or MultiCycle) and I think this is certainly worth investigating. However, I started this thread because I have been having trouble identifying polyploid cells in endoreduplicating keratinocytes and I think I may have stirred the muddy waters. I think that the problem here for flow is that we are losing the morphological information that we need to be able to say what each dot actually is. I think an LSC-based approach may be better, all I need is the time.... Derek >At 08:16 AM 12/20/99 -0600, Nick Terry wrote: >><musing quietly> >>If your hardware supports it integral versus peak is much more useful than >>width v. area. Wide beams don't help either (grateful for his 5 micron >>beam). Saving these options sometimes an extra marker (appropriate cyclins) >>can help. > >(Putting hornets back in nest - returning worms to can). Lots of mail on >this one. Sorry I should have been more specific. Peak signals can be very >helpful in doublet discrimination but only if you have narrow beam >excitation optics. IMHO adequate DNA measurements can only be made if the >cross section of your interogating beam is substantially narrower than the >diameter of the objects that you are measuring. > >Under these conditions 2 G1 cells stuck together will have a very similar >integral (yes - mostly the same as area) signal as a true single G2/M cell. >The peak signal from a G2/M cell will however be significantly greater than >that from a G1 cell and from the sequential peak signals from a G1G1 >doublet. But, the latter is only visible if the exciting beam is narrow. >In comparisons between area/width and integral (area)/peak discriminating >ability on an instrument with 5 micron excitation optics the integral peak >analysis gave the best discrimination. It is even possible to recognize >doublets that are not progressing linearly through the beam due to >turbulence or other factors. > >How anyone manages to discriminate doublets from true G2 cells with a 40 >micron beam, or nuclei with a 15-20 micron beam is beyond me. (tactfully). > >(Pondering the likelihood that there is a greater proportion of tetraploid >tumors in the literature than in reality.) Derek Davies FACS Laboratory, Imperial Cancer Research Fund, London. UK. http://www.icnet.uk/axp/facs/davies/index.html
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