Tumor dissociation

Echeagaray, Patricia L. echeagaray at sri.org
Thu Aug 27 16:42:36 EST 1998


Sigma also has a cell dissociation solution. If I remember correctly,
its formula (and most likely that of its competitors) is proprietary
(sp?). I ALSO remember being told by one of the salespeople that it was
"approximately" a 2% EDTA solution.

In regards to the initial dissociation of primary tissue, a tool I used
to use for mouse and rat livers was a "Stomacher". It was a $2,000.00
box which paddled (literally) tissues to smithereens (is this a
word?....you get the picture). You placed your tissues with a little
media into a strong baggie (made for this machine), tape it shut, put
into the machine, close it so that the top of it's door catches the top
of the bag, and turn it on.  I don't remember anyone having any trouble
with tissues treated this way, and its use was expanded to include
spleens as well.


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Patricia L. Echeagaray;  echeagaray at sri.org
Flow Cytometry Services
Southern Research Institute - Frederick
Pharmaceutical Research and Development  Dept.
431 Aviation Way     
Frederick, Maryland 21701
USA
Voice: (301) 228-2170 or (301) 694-3232 ext 111 or 276
   FAX: (301) 694-7223
echeagaray at sri.org
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>----------
>From: 	Kevin G Waddick[SMTP:waddi002 at tc.umn.edu]
>Sent: 	Tuesday, August 25, 1998 2:10 PM
>To: 	Cytometry Mailing List
>Subject: 	Tumor dissociation
>
>
>   I would appreciate it if someone would provide me with the benefit of
>their experience. When using adherent tumor cell lines, and wanting to get
>them into suspension without using trypsin, a cell dissociation solution
>from Gibco/BRL has worked well. Does anyone know the formula for this
>solution (it is not listed by Gibco/BRL). Before this solution was
>substituted,
>we used an EDTA/PIPES solution. Unfortunately, I never wrote down the
>concentrations of these chemicals used in the mixture; does anyone have a
>protocol for making and using this solution they would be willing to
>share?
>   My next question is about the best way to dissociate the cells from a
>primary patient tumor for analysis and sorting by flow cytometry. In the
>past, I used a tissue grinding pestle in a glass tube to break down organs
>ex vivo and combined this with incubation in trypsin. Does anyone know of
>a better way to do this such that degradation of cell-surface proteins by
>trypsin can be avoided (or maybe an optimal concentration and incubation
>time for trypsin that combines a good cell dissociation with minimal
>protein digestion)? Do the solutions mentioned in the previous paragraph
>work when used with solid tumors? Or are there better ways to do what I
>want to do? Whatever seems to work well for you, could you outline for me
>how it is done or provide me with a reference?
>   I would be grateful for assistance in learning this without having to
>put in a lot of time in the library.  
>
>Kevin G. Waddick, Ph.D.
>2520 Silver Lane NE, Apt. 201
>St. Anthony, MN  55421
>
>



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