Help with 3H Thymidine Assay with lymphocytes / cell harvester required?

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Tue Oct 2 20:18:24 EST 2007


Hi David,

basically,  washing them many times (I remember having washed adherent
cells in 24 well plates at least 6 times) might do the job, but it is
very likely that you increase the error in your data as you can't
control the number of cells you suck away during that procedure. Maybe
you also want to use any kinds of spin columns (maybe even a pipet
filter tip will do) that can hold back cells and let nucleotides run
through.

What I'd recommend to try is a method that currently used in my lab in
an assay where the binding of radioiodinated antibodies on cells is
measured, so the incorporated radioactivity needs to be separated from
the floating one. It's pretty simple and easy actually: the cell
suspension with the radioactive label is layered on a certain kind of
oil (see below) and briefly spun (maybe 10 seconds) just to drive the
cells through the layer. The whole thing is frozen in liquid nitrogen,
then the tube is cut in the middle of the oil (layer height: 5-10mm)
and the radioactivity in the pellet is counted.

Here comes the below: as you already imagined, the oil serves to
separate the cells from the aqueous liquid, thus the density needs to
be between that of your medium and that of the cells. We use a mix of
dodecane and bromododecane  I don't have the ratio at my hands, but I
could dig it out (means ask my student next week when she's back from
holidy).
Meanwhile also google might help you:
http://www.google.de/search?hl=de&client=firefox-a&rls=org.mozilla%3Aen-US%3Aofficial&hs=39C&q=dodecane+bromododecane+cells+insulin&btnG=Suche&meta=
But I think, the reference is this paper by Hampe et al.:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16025399

The trick is to use thin walled eppis and strong scissors (we use
chinese style, as for trimming Bonsais. Iron sheet cutting scissor
might work as well).

Best regards,

Wolfgang

On Oct 2, 9:40 pm, David Liu <aman... from yahoo.com> wrote:
> Hello,
>
> I will be doing a 3H Thymidine uptake assay on primary
> T cells for the first time.  The protocol that was
> given to me was for an adherent cell line, and I'm
> finding that many people use a vacuum cell harvester
> to wash non-adherent cells in the 3H thymidine assay.
> Unfortunately, it is difficult for me to have access
> to such an instrument, and I'm wondering if it is
> possible to do this assay without a vacuum cell
> harvester.  Does anyone have any experience with
> performing this assay on non-adherent cells without a
> harvester?  Is it possible to just centrifuge and
> pipette out supernatant to wash away unincorporated
> thymidine and TCA?  What recommendations do you have?
> Or is a cell harvester a must?
>
> Thanks,
> David
>



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