Re: Syto dyes for plant nuclei in living cells

Posted by Alison J. North on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Syto-dyes-for-plant-nuclei-in-living-cells-tp592149p592157.html

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Hi John and Tobias,

This may be a completely unhelpful response because I was not working on
plants and I was looking for dyes in the red/far red range, not blue.
However, a few years ago I tested a whole range of the red Syto dyes to
try to use them to follow cell division using live cell imaging
(together with a GFP construct) and I found something very interesting
with Syto 61.  Although I had the same problem as Tobias when I imaged
through Texas Red filter sets - i.e. the mitochondria obscured the
nuclear signal - I found that if I acquired through the far red (Cy5)
filters the signal was now predominantly nuclear.  The longer I left it,
the more the nuclear signal seemed to intensify while the remaining
mitochondrial signal soon bleached away. I called Mol. Probes to ask
them about this and they said that the dye becomes red-shifted when it's
highly concentrated - which fits with the fact that the mitotic cells
were brightest.  Anyway, unlike most of the nuclear dyes it didn't
affect cell division so I could image away for hours/days.

One slight problem - the signal and localisation appeared to be highly
cell specific.  It was great in MDCK cells but I seem to remember I
couldn't get the same effect in HeLas.  Still - might be worth a shot if
a far red dye would do?

Best wishes,
Alison


Tobias Baskin wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> John,
>         One or two years ago, we got a group of syto dyes for this very
> purpose, imaging in arabidopsis roots. We were hoping to image DNA along
> with a GFP reporter and therefore we tested sytos that have long
> wavelength emission (we were working on confocal, without ability to do
> uv excitation). None of them worked for us. They tended to be either
> toxic or dim. The other thing we noticed was that because the cells are
> crammed with mitochondria that move all over the place, it was difficult
> to follow nuclear DNA against this hive of mitochondrial motion. But we
> never tested blue ones.
>
>         Good luck,
>                 Tobias
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Do any of the plant biologists out there have experience using the
>> Syto dyes from Molecular Probes to stain nuclei of living cells.  In
>> particular, they list 5 different dyes in the blue emission range
>> (Syto 40-45).  Syto 42 looks good to me on paper.  What do you think.  
>> Thanks, John.
>> --
>> Runions signature
>> *********************************
>> C. John Runions, Ph.D.
>> School of Life Sciences
>> Oxford Brookes University
>> Oxford, UK
>> OX3 0BP
>>
>> email:  [hidden email] <mailto:[hidden email]>
>> phone: +44 (0) 1865 483 964
>> Runions' lab web site
>> <http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>
>>  
>> Visit The Illuminated Plant Cell
>> <http://www.illuminatedcell.com/ER.html> dot com
>> Oxford Brookes Master's in Bioimaging with Molecular Technology
>> <http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt>
>
>
> --
>
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--
Alison J. North, Ph.D.,
Research Assistant Professor and
Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab     ++ 212 327 7486
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