Tritiated-thymidine labelling for replication
nikolaic at VISAR.WUSTL.EDU
Sun Oct 29 13:39:36 EST 1995
> From: David Lukac <lukac at a1.mscf.upenn.edu@in>
> Subject: Tritiated-thymidine labelling for replication
> Date: 28 Oct 1995 21:11:27 GMT
> Can anyone send me a good reference for a protocol to measure
> DNA replication of cultured cells? Specifically, I need to compare
> replication of BHK-21 cells under different conditions, and I want
> to start with tritiated-thymidine incorporation.
For fast proliferating cells plate 10e4 cells/well on 96 well plate
(increase cell density up 10e6/weel if they are slow proliferating).
Use your different conditions.
After 48-72 hr add 1 uCi in 10 ul/well tritiated-thymidine
(Amersham) for another 12-18 hr.
Stop cell growth by freezing the plate at -20oC. (Plate could be stored
at this conditions for a while).
The numbers of automated or manual harvesters might be used (depends of
what available). In theory, the cells first washed off the plate into
glass fiber filters in PBS or wather. Then they are desintegrated by
washing with 5% TCA, so the only DNA remains on filter while proteins
and lypides are washed away. Subsequently filters additionaly washed
with 70% EtOH. The filters should be carefully dried (o/n under the
hood or 1-2 hr at 98oC).
Again everything depends of what type of equipment is available.
It could be either direct or liquid scintillation beta counting (refer
instructions supplied with equipment).
If direct counting, the dried filters placed directly under the stream
of particulary formulated gas and sensor meassures the radioactivity
(Packard sells such direct counters together with harvester and special
handels, counts 96 poins simultaneously)
or place the filter in tube supplied with counter, fill the tube with
appropriet coctail and count it.
More information about the Methods