Cell Culture Question

Anton Scott Goustin asg at cmb.biosci.wayne.edu
Mon Jan 29 12:54:18 EST 1996


Simon Dawson <Simon.Dawson at nott.ac.uk> wrote:
>  I have some Swiss 3T3 cells and I would like to make them go quiescent (i.e. 
>stationary phase). After all (or most anyway) of the cells appear to be 
>quiescent I would like to reinduce growth again and monitor the production of 
>certain proteins via Western blotting etc.

Simon,
This approach has been around for nearly 20 years.  You choose wisely
when you suggest Swiss 3T3: they have been used alot in this sense.
Before you do the experiment, you might look carefully at the literature
because your question may have already been posed and answered.
Different 3T3 lines (from different strains of mice) respond to growth
factor (serum) starvation differently, but this works for Swiss 3T3:
1)  See the cells at about 50% confluence and let them grow O/N in 10%
    fetal calf serum/DMEM.  In the morning, they will still be subcon-
    fluent, hopefully.
2)  Next morning, remove medium, rinse cell monolayers with Dulbecco's
    PBS, and replace medium with 0.5% FCS/DMEM.
3)  Culture 24-72 hr in this serum-reduced medium.
    Cells will go in G0 (G zero) within 16 hr.
4)  Remove medium and re-feed with 10% FCS/DMEM (or growth factors
    cocktail made up in 0.5% FCS/DMEM).
5)  Within 12-16 hr, the first cells will move into S phase, and ac-
    cumulation of cells in S phase will then continue for another 8 hr.
    Cells will continue in the cycle after that time, G2, M, etc, but
    will gradually become asychronous.
The reason for the 24-72 hr in step 3) involves your particular gene.
Some genes shut-off almost completely within 24 hr, other require more
time.  You'll have to determine this empirically, for maximal signal-
to-noise.




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