DIANE M SHARP 695-7248 wrote: > Dear colleagues, > > I have a B-D FACScan and routinely do PI staining for cell cycle analysis. I > gate on width and area (doublet discrimination). Does this guarantee > exclusion of doublets? In general, the electronic approaches to excluding aggregates (BD's Width vs Area, Coulter's Integral vs Peak, also Coulter's Time of Flight) work fairly well. I would never say they guarantee this exclusion, however. I've seen artifactual staining patterns (e.g.., with squamous cells) that can cause disproportionate signal duration leading to false detection of aggregates. It's also a problem with cells that have been treated with cycle modulators/blockers. So, in cases where doublet/aggregate detection is clearly separated, it's probably safe to trust it. In cases where resolution is diminished, or synthesis progression has been disturbed, better to be conservative with the interpretation. In my experience, the Width vs Area configuration seems to be a bit more sensitive than Integral vs Peak, although Time of Flight works well in the absence of any fluorochrome. Ultimately, it's best to 1) prevent doublets/aggregates as much as possible with modifications to your labeling protocols, and 2) validate the detection of aggregates via cell sorting and/or microscopy. An aside . . . we've used Time of Flight very effectively to improve purity in cell sorting. Alternatively, many of the DNA histogram fitting routines available have the ability to "correct" for doublets/aggregates in a sample (you may hear from them in response here). These methods are very effective as well, although less so in that they are not usable in real time. > I can clearly gate out doublets that appear to be 4N DNA content with a large > pulse width signal, but I also often see greater than 4N events that do not > have a large width pulse. Are these truly >4N cells? The cell lines I use > are adherent human tumor and fibroblast cell lines. Any input would be > appreciated. If one trusts the doublet exclusion method employed, one then must accept that cells that lie on the diagonal line with the "singlet" region must ultimately be seen as non-aggregate. This is not really a problem, since most cell lines have abnormal DNA content, and many may be bi/multiclonal. Certainly in the context of primary tissue, aneuploidy detected along with "normal" diploid cells would be seen the same way. There are also cases where cells may endoreduplicate. MAK. -- Mark A. KuKuruga, Managing Director University of Michigan Core Flow Cytometry kukuruga@medmail.med.umich.edu
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