On Wed, 17 Mar 1999, DIANE M SHARP 695-7248 wrote: > 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? 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. I think that the short answer is that it doesnt guarantee exclusion of all doublets but will certainly get rid of a lot of them from the analysis. The Area v width plot will reveal two G1 cells that travel through the beam consecutively but not two that travel side by side. Doublets will show a greater width than true G2 cells - although this is much more applicable to small round cells - epithelial cells, where there can be a degree of cytoplasmic staining, can be difficult - keratinocytes seem to be a particular problem. Looking at the plot with area on the y axis the trail of single cells travels upwards at an angle about 10 degress off the vertical (towards the top right). I usually put a polygonal gate to exclude the obvious doublets. However certain cell types will show endoreduplication and in these cases it is best to run all samples ungated and decide on gating strategies later as the multiploid cells will follow the angle of the single cell population. By excluding all cells above 4n it is possible to lose what are single cells with multiple nuclei by labelling them as clumps - a sample showing true reduplication will have peaks at 2n, 4n, 8n, 16n etc but none at 6n, 10n and other n's in between. Knowing what is happening biolgically will help you decide whether you are getting real changes or clumping as will sorting the relevant populations onto a slide and checking microscopically. Derek **************************************************************************** * 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 * ****************************************************************************
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