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Dear all,

Look at that.
http://www.biotechniques.com/news/biotechniquesNews/biotechniques-319047.ht
ml?utm_source=BioTechniques+Newsletters+%26+e-Alerts&utm_campaign=d69106c1f
1-Daily_06032011&utm_medium=email

Best

Jo

Dipl. Biol. Joachim Hehl
Staff Scientist
LMC-Light Microscopy Centre, ETH Zurich Hönggerberg
Schafmattstrasse 18, HPM F16.1
CH-8093, Zurich, Switzerland

Web: www.lmc.ethz.ch
Phone:     +41 44 633 6202
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Am 7/26/11 12:55 PM schrieb "Masur, Sandra" unter <[log in to unmask]>:

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>
>Dear all who indicate that various nuclear dyes will cause
>
>> cultured cells....to... dye (usually quite
>> spectacularly)
>
>You mean that the "dye" (addition of a color) will cause the cells to
>"die" (cease living).
>
>English spelling can be very confusing!
>
>On Jul 25, 2011, at 10:24 PM, Cameron Nowell wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Hi Paul,
>> 
>> I have used Hoechst 33342 in the past for live imaging both primary and
>> cultured cells. After about 24 hours they will dye (usually quite
>> spectacularly). But i have managed to image cells divinging with
>> Hoechst, but the best is only ever one round of division.
>> 
>> Cheers
>> 
>> Cam
>> 
>> 
>> 
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[log in to unmask]]
>> On Behalf Of Paul Rigby
>> Sent: Tuesday, 26 July 2011 11:46 AM
>> To: [log in to unmask]
>> Subject: Re: Long term Nuclear labeling in live cells
>> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Hi Leoncio,
>> While I can't suggest a solution, I am interested in your comments on
>> the dyes you have tried for your live cell imaging, specifically, the
>> UV/violet excited dyes Hoechst and DAPI.
>> 
>> My experience has been that Hoechst 33342 will stain nuclei in living
>> cells and has not proved toxic over 24 hours of imaging, albeit using
>> very low laser powers and only infrequent imaging. Were you using
>> Hoechst 33342 or Hoechst 33258 (or the slightly longer wavelength
>> excited Hoechst 34580)? Also, what concentration were you using?
>> Frigault et al (J Cell Sci, 122, 753-767, 2009) suggest that to avoid
>> toxicity effects in live cell imaging, these dyes may need to be used at
>> 10-100x more dilute concentrations than usually recommended.
>> 
>> Also you mentioned using DAPI for nuclear staining. This probe is
>> relatively live cell membrane impermeant and usually requires quite high
>> concentrations to get significant nuclear labelling in living cells. At
>> high concentrations DAPI is also toxic so I am not surprised you had
>> problems with this dye.
>> 
>> As an aside, has anyone seen inhibition of cell division when using
>> these DNA intercalating dyes? Some people suggest that cell division is
>> inhibited, but others have not reported any problems. What is the
>> concensus?
>> 
>> Regards
>> Paul
>> 
>> Assoc. Prof. Paul Rigby
>> Centre for Microscopy, Characterisation & Analysis (M510)
>> The University of Western Australia
>> 35 Stirling Highway
>> Crawley  WA  6007
>> Australia
>> 
>> 
>> 
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[log in to unmask]]
>> On Behalf Of Vergara, Leoncio A.
>> Sent: Tuesday, 26 July 2011 5:20 AM
>> To: [log in to unmask]
>> Subject: Long term Nuclear labeling in live cells
>> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> I was wondering if there is any non toxic alternative help in
>> segmentation of nucleus and cytosol compartments to measure protein
>> translocation dynamics in 12-24 hrs time lapse experiments in live
>> cultured cells. 
>> 
>> We are working with cell lines stably expressing both GFP and Strawberry
>> fluorescent protein constructs. We want to follow the single cell
>> translocation process and correlate the two signals over a period of
>> 12-24 hrs. We are looking for a tool to aid in segmentation between both
>> compartments for automated image analysis. The problem is that all the
>> DNA binding dyes we have tried are cytotoxic. We have tried DRAQ5, DAPI,
>> HOESCHT and several SITO dyes from molecular probes. We have not tried
>> the Vybrant(r) DyeCycle stains from Invitrogen, violet and Ruby could be
>> compatible with GFP and Strawberry, but a call to their technical
>> support was not very encouraging.
>> 
>> I am wondering if there are any long term live cell tracking dyes that
>> can label the cytosol and give a "negative" image of the nucleus, be non
>> toxic and be compatible with GFP and Strawberry.
>> 
>> Invitrogen also has the CellLight reagents but they are base either on
>> GFP or RFP so won't be a solution either since we are using those
>> channels. I guess we could develop a far-red construct for triple FP
>> labeling, but I was hoping for an easier solution... :)
>> 
>> For additional reference: the microscope we are using is a Prairie
>> Technologies Swept Field Confocal, we have 4 channels available (typical
>> DAPI/FITC/TRITC/Cy5) to work. This is not an spectral imaging system.
>> 
>> Thanks in advance, any suggestion would be greatly appreciated
>> 
>> Leoncio Vergara
>> Technical Director
>> Optical Microscopy Core
>> Galveston 
>> Texas
>> 
>> 
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