Hi Bill, Very interesting question! I posted something similar a year or so ago and didn't really get any convincing answers. Like you, we have seen endoreduplication but in primary foreskin keratinocytes. I have been a little concerned that with width v height/integral gating you may either lose the real reduplicated cells and the distinction between true multiploid cells and 'clumped' cells becomes blurred. By DNA flow alone I think it is difficult to truly convince yourself (or anyone else) that they are real multiploid cells. One suggestion was to use nuclei only but then if the cells are multinucleate as well as multiploid this can be counter-productive. It seems that by flow you need to add some other parameter but I am not sure what it is. I suppose you could sort the relevant popluations but this may be time consuming. My approach has been to move the assay to a static system - the Laser Scanning Cytometer - where we can relocate the reduplicated cells and confirm that they are either single large cells or multinucleated cells or simply aggregates. A further problem we encountered was the very low frequency of these cells in the real-life situation - as you have treated your cells there may well be a greater percentage to aim at. Good luck and let us know how you get on! Derek On Wed, 13 Dec 2000, Bill Telford wrote: > We have a drug that seems to induce endoreduplication in HL-60 cells i.e. > when we analyze simultaneously for DNA content and cyclin B1 expression > (with area vs. width doublet exclusion), we see a 4N subpopulation negative > for cyclin B1 expression. If we don't do area vs. width doublet > discrimination, we in fact see a chain of cell cycles extending out to the > 16N range and possibly beyond, with low-cyclin B1 subpopulations again > suggestive of endoreduplicated cells. Microscopy of these cells shows very > large cells with multiple nuclei, confirming that we probably do have cells > that have gone through several cycles of endoreduplication. > We know that we are seeing 4N endoreduplication by flow. My question > is...is it valid and possible to clearly identify 8N and 16N (or more) > endoreduplicated cells by flow while still doing doublet/clump > discrimination? My concern is that these very large cells might not be > distinguishable from doublet G2 4N, 8N etc. cells as the abnormal DNA > content and cell size increases, with both types expressing no cyclin B1. > If it is not valid to extend this endoreduplication analysis past a certain > point, would adding a G1 cyclin analysis (such as cyclin E) to the analysis > allow less ambiguous discrimination of these abnormal cells? ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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