Hi Ron, I have found that this isnt an uncommon phenomenon but one that is often overlooked. Assuming you are using pulse processing to exclude the obvious doublets quite often in established cell lines there is a re-duplicated population containing '8n' cells. Cells that are artifically blocked in G2 by, for example, nocodazole do sometimes show cells that apparently double their DNA but do not divide. If there is excessive clumping (and pulse processing will not be able to remove 100% of clumped cells), you will see peaks at the 6n, 8n, 10n and so on. Generally in reduplication, the 8n peak clearly looks like a 'real' population. Its always worth saving your data ungated and checking during analysis. We have seen in certain cell types that transfection can lead to changes in the DNA content of cells. Maybe by adding an internal control of known DNA content you can re-assure yourself that you are actually seeing an increase in DNA content rather than it being a staining artefact (although to see such a large change due to something like changes in DNA conformation might be unusual in itself). Sorting the 8n population (or using a Laser Scanning Cytometer) will allow you to see whether they are single large cells or clumps. Hope that helps, Derek On Tue, 11 Dec 2001, Ron Hill wrote: > One of our users has observed an apparent doubling in the amount of > DNA (based on PI staining) in the 2N population of NIH3T3 cells after > selection in drug to establish cell lines that stably express a gene > of interest. The cells were engineered by retrovirus. The doubling is > compared to the parental cells. The selected cells have a 4N > population with 4 times the DNA of parental 2N cells. Has anyone ever > seen this. Is it likely to be some artifact of staining? ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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