Thank you very much to all of you for your comments.
Sorry that I did not reply to some of you. We did not get a grant and
have to stop doing live imaging.
Thanks,
Olga
Olga Makarova, PhD
Research Specialist
5323 MSRB III
>>> Olga Makarova <[log in to unmask]> 8/9/2010 12:53 PM >>>
Thank you , Tom,
How come CO2 will penetrate through mineral oil?
How quickly DNA dye effect mitosis?
Does histone- fluorescent proteins available commercially? Do you mean
to cotransfect histone- fluorescent proteins DNA along with my tagged
protein?
Olga
Olga Makarova, PhD
Research Specialist
5323 MSRB III
>>> Artem Pliss <[log in to unmask]> 8/6/2010 5:53 PM >>>
Hi Olga,
You may apply a drop of mineral oil on top of your medium- this would
reduce
the evaporation. For live cells DNA stain most people use Hoechst
33342, you
may also refer to this article: Martin et al (2005) DNA labeling in
living
cells. Cytometry A 67:45–52. In my experience long term applications
of any
DNA dye reduces the cell viability and may prevent them from dividing.
As an
alternative you may consider histone- fluorescent proteins fusion.
Best,
Art
* *Regarding the DNA dye Hoechst usually works well- but
On Fri, Aug 6, 2010 at 2:10 PM, Olga Makarova
<[log in to unmask]>wrote:
> Hi everybody,
>
> I am trying to use DeltaVision environmental chamber 40X, time lapse
to
> figure out localization of my EYFR-tagged protein. I did it once.
> When I fixed cells , endogenous protein predominantly is in the
nucleus
> and also in cytoplasm, but sometimes only in cytoplasm.
> EGFP-tagged also shows the same pattern. I thought that transition
could
> depend on stage of cells, particularly mitosis.
> I am going to include DIC imaging. I also want to add DNA dye for
the
> living cells. What would be the best choice? Or are there any other
dyes to
> track different stages of cells?
> I am using transiently transfected HPV epithelial cells which
divided
once
> in 4 days. Probably I need some synchronization before or after
> transfection. What would be a better choice.
> Also I found that media evaporated rather quickly in the chamber
making
> media ingredients more concentrated. Any advice regarding that
issue?
> Thank you very much for any advise.
>
> Olga
>
>
>
> Olga Makarova, PhD
> Research Specialist
> 5323 MSRB III
>
>
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