Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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Grant MacGregor |
Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
We have two LSM510 META's, neither of which is equipped with a UV laser. On one of the 510 META's, users currently employ a TiSa multi-photon laser to excite DAPI and Hoechst and activate GFP's etc, but that laser is going to be moving to a new system. Hence, we are looking for advice on dyes with which to counter stain nuclei, which don't require use of a laser in the UV range.
Our users employ different species (fly, fish, mammalian cells and tissues) and all the usual xFP's (C-,Y-, G- and mCherry). From what I've seen in the literature and at meetings, one of the most popular dyes appears to be DRAQ5. This emits in the far-red, so it can easily be resolved from the other reporters. It's also relatively photo stable and live cell-permeant. Does anyone have advice on alternative dyes that might be more versatile than DRAQ5 for such purposes ? Ideally, to save money and improve quality of reagents, we'd like to develop a system of having our core supply investigators with aliquots of nucleic acid stains and secondary antibodies, which we know will be robust for use in analysis with our core's instrumentation. Thanks for any advice you can offer on this matter. Grant MacGregor D. Phil., Associate Director, Optical Biology Core, Developmental Biology Center Associate Professor, Department of Developmental and Cell Biology, Center for Molecular & Mitochondrial Medicine & Genetics, Developmental Biology Center, University of California, Irvine 2042 Hewitt Hall Irvine, CA 92697-3940 |
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Joachim Hehl |
Re: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal hi, try the Vybrant Dye Cycles from invitrogene (no commercial interest) Jo -----Original Message----- From: Confocal Microscopy List on behalf of Grant MacGregor Sent: Wed 5/28/2008 4:00 AM To: [hidden email] Subject: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ? Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have two LSM510 META's, neither of which is equipped with a UV laser. On one of the 510 META's, users currently employ a TiSa multi- photon laser to excite DAPI and Hoechst and activate GFP's etc, but that laser is going to be moving to a new system. Hence, we are looking for advice on dyes with which to counter stain nuclei, which don't require use of a laser in the UV range. Our users employ different species (fly, fish, mammalian cells and tissues) and all the usual xFP's (C-,Y-, G- and mCherry). From what I've seen in the literature and at meetings, one of the most popular dyes appears to be DRAQ5. This emits in the far-red, so it can easily be resolved from the other reporters. It's also relatively photo stable and live cell-permeant. Does anyone have advice on alternative dyes that might be more versatile than DRAQ5 for such purposes ? Ideally, to save money and improve quality of reagents, we'd like to develop a system of having our core supply investigators with aliquots of nucleic acid stains and secondary antibodies, which we know will be robust for use in analysis with our core's instrumentation. Thanks for any advice you can offer on this matter. Grant MacGregor D. Phil., Associate Director, Optical Biology Core, Developmental Biology Center Associate Professor, Department of Developmental and Cell Biology, Center for Molecular & Mitochondrial Medicine & Genetics, Developmental Biology Center, University of California, Irvine 2042 Hewitt Hall Irvine, CA 92697-3940 |
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Nuno Moreno |
Re: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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In reply to this post by Grant MacGregor
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal olha outros. Grant MacGregor wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have two > LSM510 META's, neither of which is equipped with a UV laser. On one of > the 510 META's, users currently employ a TiSa multi-photon laser to > excite DAPI and Hoechst and activate GFP's etc, but that laser is going > to be moving to a new system. Hence, we are looking for advice on dyes > with which to counter stain nuclei, which don't require use of a laser > in the UV range. > > Our users employ different species (fly, fish, mammalian cells and > tissues) and all the usual xFP's (C-,Y-, G- and mCherry). From what > I've seen in the literature and at meetings, one of the most popular > dyes appears to be DRAQ5. This emits in the far-red, so it can easily > be resolved from the other reporters. It's also relatively photo stable > and live cell-permeant. > > Does anyone have advice on alternative dyes that might be more versatile > than DRAQ5 for such purposes ? Ideally, to save money and improve > quality of reagents, we'd like to develop a system of having our core > supply investigators with aliquots of nucleic acid stains > and secondary antibodies, which we know will be robust for use in > analysis with our core's instrumentation. > > Thanks for any advice you can offer on this matter. > > > Grant MacGregor D. Phil., > > Associate Director, > Optical Biology Core, Developmental Biology Center > > Associate Professor, > Department of Developmental and Cell Biology, > Center for Molecular & Mitochondrial Medicine & Genetics, > Developmental Biology Center, > > University of California, Irvine > 2042 Hewitt Hall > Irvine, CA 92697-3940 > > > > |
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Tobias Baskin |
Re: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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In reply to this post by Grant MacGregor
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Greetings I don't know off hand the tech specs of your instruments but I wanted to mention that DAPI can be nicely excited with a 405 nm laser line. You don't need UV. Tobias Baskin >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have two >LSM510 META's, neither of which is equipped with a UV laser. On one >of the 510 META's, users currently employ a TiSa multi-photon laser >to excite DAPI and Hoechst and activate GFP's etc, but that laser is >going to be moving to a new system. Hence, we are looking for >advice on dyes with which to counter stain nuclei, which don't >require use of a laser in the UV range. > >Our users employ different species (fly, fish, mammalian cells and >tissues) and all the usual xFP's (C-,Y-, G- and mCherry). From what >I've seen in the literature and at meetings, one of the most popular >dyes appears to be DRAQ5. This emits in the far-red, so it can >easily be resolved from the other reporters. It's also relatively >photo stable and live cell-permeant. > >Does anyone have advice on alternative dyes that might be more >versatile than DRAQ5 for such purposes ? Ideally, to save money and >improve quality of reagents, we'd like to develop a system of having >our core supply investigators with aliquots of nucleic acid stains >and secondary antibodies, which we know will be robust for use in >analysis with our core's instrumentation. > >Thanks for any advice you can offer on this matter. > > >Grant MacGregor D. Phil., > >Associate Director, >Optical Biology Core, Developmental Biology Center > >Associate Professor, >Department of Developmental and Cell Biology, >Center for Molecular & Mitochondrial Medicine & Genetics, >Developmental Biology Center, > >University of California, Irvine >2042 Hewitt Hall >Irvine, CA 92697-3940 -- _ ____ __ ____ / \ / / \ / \ \ Tobias I. Baskin / / / / \ \ \ Biology Department /_ / __ /__ \ \ \__ 611 N. Pleasant St. / / / \ \ \ University of Massachusetts / / / \ \ \ Amherst, MA, 01003 / / ___ / \ \__/ \ ____ http://www.bio.umass.edu/biology/baskin/ Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243 |
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Jennifer Waters |
Re: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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In reply to this post by Grant MacGregor
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Our users use TOPRO3 from Invitrogen.
On Wed, May 28, 2008 at 9:07 AM, Tobias Baskin <[hidden email]> wrote: Greetings -- Jennifer Waters, Ph.D. Director, Nikon Imaging Center at Harvard Medical School |
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Glen MacDonald-2 |
Re: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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In reply to this post by Grant MacGregor
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Fixed or live tissues? To-Pro-3 is membrane impermeant to most live cell types, but labels well after fixation. Use a high concentration to account for its bleaching. You may want to avoid Alexa594 in combination since it excites with 633-638 nm lasers sufficiently to appear in the far red channel. regards, Glen Glen MacDonald Core for Communication Research Virginia Merrill Bloedel Hearing Research Center Box 357923 University of Washington Seattle, WA 98195-7923 USA (206) 616-4156 [hidden email] ************************************************************************ ****** The box said "Requires WindowsXP or better", so I bought a Macintosh. ************************************************************************ ****** On May 27, 2008, at 7:00 PM, Grant MacGregor wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- > bin/wa?S1=confocal We have two LSM510 META's, neither of which is > equipped with a UV laser. On one of the 510 META's, users > currently employ a TiSa multi-photon laser to excite DAPI and > Hoechst and activate GFP's etc, but that laser is going to be > moving to a new system. Hence, we are looking for advice on dyes > with which to counter stain nuclei, which don't require use of a > laser in the UV range. > > Our users employ different species (fly, fish, mammalian cells and > tissues) and all the usual xFP's (C-,Y-, G- and mCherry). From > what I've seen in the literature and at meetings, one of the most > popular dyes appears to be DRAQ5. This emits in the far-red, so it > can easily be resolved from the other reporters. It's also > relatively photo stable and live cell-permeant. > > Does anyone have advice on alternative dyes that might be more > versatile than DRAQ5 for such purposes ? Ideally, to save money and > improve quality of reagents, we'd like to develop a system of > having our core supply investigators with aliquots of nucleic acid > stains and secondary antibodies, which we know will be robust for > use in analysis with our core's instrumentation. > > Thanks for any advice you can offer on this matter. > > > Grant MacGregor D. Phil., > > Associate Director, > Optical Biology Core, Developmental Biology Center > > Associate Professor, > Department of Developmental and Cell Biology, > Center for Molecular & Mitochondrial Medicine & Genetics, > Developmental Biology Center, > > University of California, Irvine > 2042 Hewitt Hall > Irvine, CA 92697-3940 > > > > |
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Jean Brennan |
Re: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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In reply to this post by Grant MacGregor
DRAQ5 works just like DAPI on but emits in far-red so it's nice for confocal systems that doesn't have a UV laser. The staining with DRAQ5 is as easy as DAPI and the dilution is 1:5000. It was developed for the FACScan so it can be exited by a 488 laser and still emit over 633. If you want to use more channels above 600 it's very difficult to subtract the DRAQ5 signal. To-Pro 3 has a very tight emittion spectrum and would allow you to use 680 and about with it. I have used To-pro 3 with fixed cells only.
Hope this helps.
Jean
Jean Brennan
Research Specialist Rothstein lab, Dept of Neurology Johns Hopkins University 600 N. Wolfe St., Meyer 6-174 Baltimore, MD 21287 410-614-4119; FAX: 410-955-0672 [hidden email] >>> Grant MacGregor <[hidden email]> 05/27/08 10:00 PM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal We have two LSM510 META's, neither of which is equipped with a UV laser. On one of the 510 META's, users currently employ a TiSa multi-photon laser to excite DAPI and Hoechst and activate GFP's etc, but that laser is going to be moving to a new system. Hence, we are looking for advice on dyes with which to counter stain nuclei, which don't require use of a laser in the UV range.
Our users employ different species (fly, fish, mammalian cells and tissues) and all the usual xFP's (C-,Y-, G- and mCherry). From what I've seen in the literature and at meetings, one of the most popular dyes appears to be DRAQ5. This emits in the far-red, so it can easily be resolved from the other reporters. It's also relatively photo stable and live cell-permeant.
Does anyone have advice on alternative dyes that might be more versatile than DRAQ5 for such purposes ? Ideally, to save money and improve quality of reagents, we'd like to develop a system of having our core supply investigators with aliquots of nucleic acid stains and secondary antibodies, which we know will be robust for use in analysis with our core's instrumentation.
Thanks for any advice you can offer on this matter.
Grant MacGregor D. Phil.,
Associate Director,
Optical Biology Core, Developmental Biology Center
Associate Professor,
Department of Developmental and Cell Biology,
Center for Molecular & Mitochondrial Medicine & Genetics,
Developmental Biology Center,
University of California, Irvine
2042 Hewitt Hall
Irvine, CA 92697-3940
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Bruno Saubaméa |
Re: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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In reply to this post by Grant MacGregor
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi,
we routinely use TO-PRO 3
(Invitrogen). It gives a strong staining of DNA with no staining of RNA and is
therefore perfect for nucleus counterstaining. On the contrary TOTO3 stains RNA
(although less than DNA) and requires a RNAse treatment for good results. Also
TOPRO3 (~280 euros/ml) is far cheaper than TOTO3 (~500 euros/ml).
We use it a a concentration of
1/1000 (10 min for adherent cells, 20 min for thick tissue sections) in PBS.
Take care to (1) not overrinse after staining (2x5 min is enough) since TOPRO3
can be washed out and (2) image it with a low laser intensity (10% of 633nm
laser line on a leica TCS SP2) to prevent photobleaching.
Best regards
Bruno
Bruno SAUBAMEA
EA 3621 & Service Commun d'Imagerie Cellulaire et Moléculaire
Faculté des Sciences Pharmaceutiques et Biologiques
Université Paris Descartes 4, avenue de l'Observatoire 75006 PARIS tel : 01.53.73.97.13
fax : 01.53.73.99.09
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Luis A. Rivera |
Re: Anything better or more versatile than DRAQ5 for imaging of nuclei without short wavelength laser ?
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In reply to this post by Tobias Baskin
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal June 2, 2008 Hi! Topias and Members: I have read from the Bioprobes 52 Newsletter from Molecular Probes that their proprietary Vybrant DyeCycle stains can be used for quantification in flow cytometry when dyes that bind to DNA in a stoichiometric manner are required.The Vybrant DyeCYcle stains are DNA-selective, cell membrane-permeant dyes that show greatly enhanced fluorescence when bound to DNA and are available in versions that can excited by 405, 488, or 532 nm lasers as they claim. Their catalog numbers are V35003, V35004, and V35005, respectively. Reference provided by Molecular Probes is Telford, William G et al. (2007) Stem CElls (in press). I am planning to try them for the first time. Luis A. Rivera, PhD Laboratory of Organic Chemistry, Photochemistry, and Nanobiotechnology University of Puerto Rico at Mayaguez Puerto Rico, USA --- Tobias Baskin <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Greetings > > I don't know off hand the tech specs of your > instruments but I wanted > to mention that DAPI can be nicely excited with a > 405 nm laser line. > You don't need UV. > Tobias Baskin > > > > >Search the CONFOCAL archive at > >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > We have two > >LSM510 META's, neither of which is equipped with a > UV laser. On one > >of the 510 META's, users currently employ a TiSa > multi-photon laser > >to excite DAPI and Hoechst and activate GFP's etc, > but that laser is > >going to be moving to a new system. Hence, we are > looking for > >advice on dyes with which to counter stain nuclei, > which don't > >require use of a laser in the UV range. > > > >Our users employ different species (fly, fish, > mammalian cells and > >tissues) and all the usual xFP's (C-,Y-, G- and > mCherry). From what > >I've seen in the literature and at meetings, one of > the most popular > >dyes appears to be DRAQ5. This emits in the > far-red, so it can > >easily be resolved from the other reporters. It's > also relatively > >photo stable and live cell-permeant. > > > >Does anyone have advice on alternative dyes that > might be more > >versatile than DRAQ5 for such purposes ? Ideally, > to save money and > >improve quality of reagents, we'd like to develop a > system of having > >our core supply investigators with aliquots of > nucleic acid stains > >and secondary antibodies, which we know will be > robust for use in > >analysis with our core's instrumentation. > > > >Thanks for any advice you can offer on this matter. > > > > > >Grant MacGregor D. Phil., > > > >Associate Director, > >Optical Biology Core, Developmental Biology Center > > > >Associate Professor, > >Department of Developmental and Cell Biology, > >Center for Molecular & Mitochondrial Medicine & > Genetics, > >Developmental Biology Center, > > > >University of California, Irvine > >2042 Hewitt Hall > >Irvine, CA 92697-3940 > > > -- > _ ____ __ ____ > / \ / / \ / \ \ Tobias > I. Baskin > / / / / \ \ \ > Biology Department > /_ / __ /__ \ \ \__ 611 N. > Pleasant St. > / / / \ \ \ > University of Massachusetts > / / / \ \ \ > Amherst, MA, 01003 > / / ___ / \ \__/ \ ____ > http://www.bio.umass.edu/biology/baskin/ > Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243 > |
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