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,
Frank






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