This group of cells are clumps of two cells. On your data plot there are also clumps of three cells at the 6N position. The advantage of the DNA area vs. width plot is the ability to gate these clumps out of your analysis excluding them from the G2M cluster. However, the increased width measurement depends on the cells lining up as they traverse the laser beam. In general this works due to hydrodynamic focusing of the sample stream inside of the sheath. However, it is not 100% effective. There will still be some cell clumps that tumble and present only one cell width as they cross the laser instead of two and these will remain in the G2M cluster. This is described in "Clinical Flow Cytometry" edited by Bauer, Duque and Shankey. Best regards, Tony Bakke Antony C. Bakke, Ph.D. Director, Special Immunology and Flow Cytometry Oregon Health Sciences University Portland, OR (503) 494-4687 >>> Robert Walt St. George Fisher <fisher@radonc.unc.edu> 01/11 3:52 PM >>> Hi. I'm somewhat of a beginner at flow cytometry, so I'd like to post a question to the group. I have noticed in some of my samples a subpopulation of cells that I can't figure out. I'm doing a PI vs. BrdU incorporation analysis on whole, EtOH fixed epithelial cells, and the population in question shows up in the DNA area vs. DNA width diagram. It has 4N DNA content, but increased DNA width so it runs above the G2/M population in this diagram. Would senescent cells show up in this area, since they tend to get larger and flatten out? If you gate this subpopulation to a DNA area vs. BrdU fluorescence diagram, they have the same BrdU fluorescence as G2/M cells. Go to this URL to see what I'm talking about: http://www.radonc.unc.edu/~fisher/flow.gif I'm open to suggestions as to what this population represents.... Thank you. Robert Fisher fisher@radonc.unc.edu
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