Hi all, This may seem a simple question and one that I feel that I already ought to know the answer to. When doing DNA analysis by flow it is generally accepted, is it not, that some form of pulse processing be used to exclude G1 doublets. On my BD machines I generally use a Width v area scattergram to define these and exclude. Normally when using nice round cells like lymphocytes or thymocytes, it is straightforward to see where the G2 population ends and the doublets begin. However, when using larger, epithelial cells, especially keratinocytes, the distinction is nowhere near as obvious. Where we are having problems is in keratinocytes where, for whatever reason, their mitosis is uncoupled from their DNA replication and they form 8n cells. My inclination would be to set a gate that runs parallel to the median of the G1 and G2 populations and extend it upwards to include potential polyploid cells. The worry is though that there may well be an increase in pulse width in these cells that further blurs the boundary of true single cells and clumps. Is there any consensus about how to analyse these sorts of plots? I generally now collect samples ungated and play around during the analysis stages. However I think that it may be difficult to estimate the true polyploid cells. I have tried analysing some of these samples on a Laser Scanning Cytometer but the distinction between clumps and polyploid and/or multinucleate cells is also not clear. Anyone have similar problems or any ideas? 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 In tenebris lux *************************************************************************
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