Fixed and permeabilized Cells and PI

Derek Davies derek.davies at cancer.org.uk
Tue Jul 20 04:01:32 EST 2004


Hi Joan,

A couple of questions need answering here - what is the fix and what 
is the perm? Good DNA CVs can be obtained after alcohol fixation or 
after detergent treatment but each should be optimised for the cell 
type. Fix and Perm reagents are generally a combination of 
paraformaldehyde and then detergent which will certainly allow PI (or 
other DNA-binding dye) to get into the cells but may not give 
brilliant CVs (as paraformaldehyde is a cross-linking fix, the 
accessibility of the dye to the DNA isn't quite as good as if a 
coagulant fixative is used. We have achieved excellent DNA profiles 
with a combination of paraformaldehyde and saponin so it is possible 
that your user will get lucky but there may be some optimisiation 
required. I would give it a go with the caveat that you cant expect 
great results from possibly suboptimal preparation.

Derek



>I have a client who has changed her mind mid experiment.  She has a 
>batch of fixed and permeabilized cells. Before she did this she used 
>PI as a viability stain.  In hindsight she would like to have done 
>cell cycle analysis.  Will adding	PI to the fixed and 
>permeabilized cells now achieve this?	Isn't that in effect what 
>Vindelov's does?  Fix, permeabilize and stain with PI?
>	 Just wondering.  If the theory is correct, it might be worth 
>her trying to do it.
>	 Thanks in advance for all thoughts on this.
>	 Joan K.



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Derek Davies					Voice: (44) 020 7269 3394
FACS Laboratory,			FAX: (44) 020 7269 3479
London Research Institute,		e_mail: derek.davies at cancer.org.uk
Cancer Research UK		mobile: 07790 604112
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Web Page: http://science.cancerresearchuk.org/sci/facs/

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