GFP and cell cycle

Arnold Pizzey a.pizzey at
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.
>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.



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

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

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