Help with 3H Thymidine Assay with lymphocytes / cell harvesterrequired?

Deitiker, Philip R via methods%40net.bio.net (by pdeitik from bcm.tmc.edu)
Wed Oct 3 12:07:08 EST 2007


 
Hi,

 We do extensive T-lymphocyte assays in our laboratory. The assay for Thymidine is
using a scintillation (beta) counter requires that the filters be dissolved in scintillation cocktail. The imprint left by the apparatus makes it easy to extract the filter circles used in the assay. 

  You can create a make shift device for harvesting cells but the problem is that you want more or less uniform dissolution of the filter matrix in the scintillation cocktail which means you don't want a large variance in the filter being added. 
It will take you a long time to harvest a single 96 well plate. 

You might see if anyone at your college has a cell harvester. 

Adherant cells will not be removed by a cell harvester, you may first have to treat them with a protease first to make them non-adherant. 

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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