*****
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
This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.
|
