GFP and cell cycle

Arnold Pizzey a.pizzey at ucl.ac.uk
Wed Oct 4 03:45:39 EST 2000


>
>I have a client who wants to do cell cylce on GFP transfected cells.  Since
>I do not have a sorter, I need to be able to do this on a Calibur.  She has
>found some comments that claim you can do DNA with a formaldehyde fix
>rather than ethanol. Is that true?  Also, I read on this list a while back
>that PI may be so bright that it may overwhelm low GFP signal so 7AAD would
>be the better choice.  I have not been able to find a protocol for cell
>cycle with 7AAD.  Any help would be greatly appreciated, as always.
>
>Thanks,
>Candace Enockson
>Medical University of South Carolina

Greetings candace,

Fix your cells in 1% formaldehyde overnight at 4'C prior to staining with
50ug/ml PI, 500ug/ml RNAase
if you can leave the cells longer in the fixative (>2days the DNA profiles
improve somewhat)

I have used this method to get cell cycle/GFP data on herpes transduced
CD34+ cells (see Gene Ther 1998 May;5(5):718-22)

I don't recall there being any problems of signal crossover between GFP/PI
that could not be fixed by dialling in modest amounts of compensation.

regards,

Arnold

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	Arnold Richard Pizzey
	Department of Haematology
	Royal Free and University College London Medical School
	98 Chenies Mews
	London WC1E 6HX
	U.K

	voice:	+44 020-7679-6234
	Fax:	+44 020-7679-6222
	email:	a.pizzey at ucl.ac.uk
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