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Hi Leonico (and Paul for the Hoechst bit)

- You can stain the nuclear envelope using FP-tagged lamins
- FP labelled histone was already suggested
- From my experience from many moons ago: You can use Hoechst (or other dyes - preferably non-intercalating ones), but you will need to dilute down as far as possible. The manufacturer will suggest concentrations that give you insanely high fluorescence. The cells will die even without the imaging procedure within 24 hr. 
Consider and try this: The cells will take up dye until binding is saturated. To obtain a concentration suitable for long term imaging, aim for submaximal staining. Do the following: Determine the number of cells per dish/well as well as the volume of culturing medium, and from this calculate the amount of dye per cell when using the concentration suggested by the manufacturer (e.g. in ug or picograms PER CELL - DO NOT USE ug/ml OR uM AS UNITS). Do serial dilutions of the dye by a factor of ten, down to about 1:100,000 and keep all your culture dishes in the incubator (no imaging yet). Check your cells after 12h, 24h, 36h, 48h for apparent death using a std tissue culture microscope. For conditions at which the cells don't die, check whether the fluorescence signal is still strong enough for imaging. Go for the lowest concentration possible. You usually would end up with less than 1 pg/cell. With this you should be able to capture images for a long time without killing the cells (I did 3 min temp resolution w/o problems). At such low dye concentration it may take up to 1-2 h for the dye to bind and the stain to fully materialise. In addition, you can reduce phototoxicity by limiting the photon flux during excitation (i.e. longer exposure time with more neutral density filters is less damaging than short exposure times with high excitation intensities). Also, if possible, you can try to red-shift your excitation and see whether you still get enough signal. 

Good luck.

Markus

Dr. rer. nat. Markus Rehm
Biomedical Research Lecturer
Dept. of Physiology & Medical Physics
& Centre for Human Proteomics and Medical Systems Biology
Royal College of Surgeons in Ireland
RCSI York House
York Street
Dublin 2
Ireland
 
phone: 00353 (0)1 4028563
email: [log in to unmask]
web: https://research1.rcsi.ie/pi/mrehm/

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Hehl Joachim
Sent: 26 July 2011 15:15
To: [log in to unmask]
Subject: Re: Long term Nuclear labeling in live cells - DYE vs DIE

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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
Natel:     +41 44 658 1679
Fax:       +41 44 632 1298
e-mail: [log in to unmask]




Am 7/26/11 12:55 PM schrieb "Masur, Sandra" unter <[log in to unmask]>:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>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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