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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:

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> 
> 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
> 
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> 
> 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
> 
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> 
> 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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