Re: prep and storage of plueral and ascitic fluid
Becky Bonner (rbonner@REX.RE.uokhsc.edu)
Wed, 8 Nov 95 07:43:44 -0800
We have been collecting effusions for about 5 or 6 years now. We collect
them in sodium heparin - as recommended by "Cytopreparatory Proceedures",
Kathy Keebler ed. The collected fluid is an excellent tissue culture medium
for cells and they are usually quite happy at room temp until transport. We
wash them 3 times in PBS and freeze them in alliquots until ready to run.
It is important that you wash all the protein out of the cell fluid as you
would any tissue culture cells. These proteins will precipitate in alcohol
and will produce nonspecific fluorescence that will play mucky muck with
your assays. Your biggest problem is that the cancer cells like to grow in
3D balls of cells that are difficult to separate into single cells. If you
have some ideas to help us with that problem - I would love to hear about
it. We syringe them with an 18 or 22 gua needle prior to freezing to help
disperse them but usually still have a lot left in fragments. Hope this helps.
Becky Bonner
QFIA Lab OUHSC
Becky-Bonner@uokhsc.edu
At 03:07 PM 11/6/95 -0500, kweber@vs4.im.med.umich.edu wrote:
>Hello, I have yet another question from the frozen expanses of Michigan
>
>We have a user that will be obtaining plueral and ascitic fluids from patients
>with malignant breast cancer that she would like to isolate the cells from and
>then run them on flow for DNA content/ apoptosis. She would like to know what
>would be the best way to draw this fluid ie, straight or with some anti-
>coagulant, how to store the fluid between the time that it is drawn and
prepped,
>and any other tricks that others more experienced may be aware of.
>
>Thanks again for all the help
>
>Kris
>
>
>
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