3H Thym incoproration assay?

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Wed May 31 02:41:14 EST 2000

Will wrote:
> Hello all,
>     I know this is real base question, but here goes anyway. Does
> anybody have a nice easy protocol for 3H thymidine incorporation into
> monolayer cells. I've looked around and can't find the info I need.
> Most of it is straight forward, my only questions are; a) should I lyse
> my cells before TCA precipitation (ie can I wash excess 3H Thymidine and
> precipitate on the plate before lysis? b) if not, is a standard SDS
> lysis buffer O.K. for running the precipitation? Thanx in advance for
> any help.
> Will
> remove X's for email response

Hi Will,
That´s the protocol we use successfully:
* grow monolayer cells on 6well dishes
* remove medium, replace by medium plus 3H-thymidine at a final
concentration of 0.5-2uCi/ml. We use 2ml per well
* incubate for the desired time (for simple proliferation assay,
incubate 12h; for cell cycle assays, pulse for no longer than 0.5-1h)
* remove medium by aspiration, discard into radioactive waste
* wash cells on plate, at least twice, with PBS (room temperature)
* drain cells, then add PBS plus 0.5% SDS (room temperature)
* let stand for 10-15min for cell lysis
* with a 1ml pipette (blue tip), pipette the viscous solution up and
down several times, then transfer the lysate to a polypropylene tube.
* wash the wells at least once with PBS/SDS, and pool the lysates
* fill to 5ml with water, vortex vigorously to shear DNA.
* the final diluted sample must have low viscosity to allow for
reproducible counting
* take two 1ml aliquots, add 200ul of 100% TCA, mix well, and let stand
on ice for 30min
* filter through Millipore GF/C glass fiber filters (prewetted with 10% TCA)
* wash twice with at least 10ml of 10% TCA, then once with Methanol
* air-dry filters, then put into scintillation vials, add 3ml of
scintillation liquid, and count.
* duplicate assays should not vary by more than 10%. If they do, the
most likely reason is that the diluted lysate is not homogenous. If that
is the case, sonication usually helps.

Best luck for your experiments,

More information about the Methods mailing list