Hello all... 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? Thanks in advance for any advice! Bill Telford DCS-NCI-NIH
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