thymidine uptake assay details or a good site? - (Dec/11/2006 )

I was looking around for a good description of a thymidine (H-3) uptake assay but haven't had much luck finding one describing the theory and explanation of results. I know a thymidine uptake assay has been done before on these same types of cells (MCF-7) with the drug our lab is currently studying (fluvastatin) but I'm wondering what EXACTLY does it measure? Does it show growth or the inhibition of it? Does it show apoptosis directly or indirectly?

I assume from my knowledge so far that live, actively proliferating cells will take up thymidine as needed for nucleotide metabolism/use, so does it only measure basically cells that are proliferating? Can I assume from the old data that decrease would mean inhibition of growth? Is it safe to assume the compound can induce apoptosis from this assay?

Thanks for any clarification or good sites explaining the theory if you have any.

Hi,

Thymidine uptake assay is a measure of cell prolliferation. 3H-Thymidine will get incorporated in to DNA of actively dividing cells and you will see difference of prolliferation between your control cells and treated cells; based on that you can calculate anti-proliferation activity or cell growth inhibition activity of a test compound.

However this assay is not a measure of apoptosis. It is likely that if you have a compound which has anti-prolliferation activity can induce apoptosis and for studying apoptosis you can do caspase (Caspase-3 is imp) assays, westerns for caspases, PARP, cytochrome c or you can do FACS analysis (PI staining), DNA ladder assay, Annexin-V assay etc. There are many.

Hope this helps.

Hi,

Thymidine uptake assay is a measure of cell prolliferation. 3H-Thymidine will get incorporated in to DNA of actively dividing cells and you will see difference of prolliferation between your control cells and treated cells; based on that you can calculate anti-proliferation activity or cell growth inhibition activity of a test compound.

However this assay is not a measure of apoptosis. It is likely that if you have a compound which has anti-prolliferation activity can induce apoptosis and for studying apoptosis you can do caspase (Caspase-3 is imp) assays, westerns for caspases, PARP, cytochrome c or you can do FACS analysis (PI staining), DNA ladder assay, Annexin-V assay etc. There are many.

Hope this helps.

Ah, ok. Thanks a lot. One more thing though... how quantitative is it and how accurate a reflection of what's going on is it? Are there any situations in which a thymidine uptake assay won't work? For example, perhaps if the cells are dying a certain way it won't work? Perhaps the cells would metabolize it or take it up differently if something happened to change their metabolism besides proliferation or death?

Probably if cells are arested in one phase of cell cycle, you won't see thymidine uptake.