The shortest path to getting a histone or nuclear targeted FP-fusion into your cells would be to use the CellularLights products from invitrogen/Life Technologies.  These are BacMan viruses encoding fluorescent proteins tagged with various targeting sequences designed to put the protein into particular cellular compartments or locations.  The BacMam baculovirus system is used for delivery, so no engineering or molecular biology required.

Being a virus system some cell types may have different transfection efficiencies but check the Invitrogen website for more details.

Basically add the supplied virus suspension to your cell culture and within 24 hrs your cells will be expressing the marker you desire.

Ian Clements
Product Manager – DeltaVision|OMX® Systems

-----Original Message-----
From: Olga Makarova [mailto:[log in to unmask]]
Sent: Monday, August 09, 2010 9:54 AM
To: [log in to unmask]
Subject: Re: DeltaVision, mitosis, EYFP-tagged protein

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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> used for urgent or sensitive issues
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