DNA dyes
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, What is the consensus on using either DAPI or Hoechst staining on live cells? Are there effects on mitosis and/or chromosome movement? Thanks, Carl Carl A. Boswell, Ph.D. Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hey Carl, I stained human mesenchymal stem cells with Hoech 33342. I then imaged them with time lapse microscopy. The cells did not die and the nuclei stained really well. However, the cells did not divide. charlie Quoting Carl Boswell <[hidden email]>: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear all, > What is the consensus on using either DAPI or Hoechst staining on live > cells? Are there effects on mitosis and/or chromosome movement? > Thanks, > > Carl > > Carl A. Boswell, Ph.D. > Molecular and Cellular Biology > University of Arizona > 520-954-7053 > FAX 520-621-3709 |
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In reply to this post by Boswell, Carl A - (cboswell)
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Carl, Charles, The response Charles described is to be expected using Hoechst, if UV (=< 405 nm) excitation is used. Same goes for DAPI, with the additional problem that DAPI requires permeabilized cells making it unsuitable for most live cell experiments. I have not tried this but I am sure someone out there probably has, namely, multi-photon confocal either 2 P or 3 P to excite Hoechst, which is PM permeable. UV by itself will elicit radical generation and strand breaking. Hoechst being a minor groove DNA dye could lead to a lower risk of scission when using 2 P or 3 P to get excitation, and might pose less risk of collateral DNA damage including unwanted crosslinking. Further, I would be interested to know whether Hoechst added to cells in culture with no light excitation and maintained through what would be a couple of passages would permit any cell division. I.E., does the dye+light abolish cell division or dye by itself? Regards All, Mario PS I know that MP illumination can really cook cells, too. Guys what about using DRAQ5 to watch mitosis? >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Dear all, >What is the consensus on using either DAPI or Hoechst staining on >live cells? Are there effects on mitosis and/or chromosome movement? >Thanks, > >Carl > >Carl A. Boswell, Ph.D. >Molecular and Cellular Biology >University of Arizona >520-954-7053 >FAX 520-621-3709 -- ________________________________________________________________________________ Mario M. Moronne, Ph.D. cell (510) 367-8497 [hidden email] [hidden email] [hidden email] |
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In reply to this post by Boswell, Carl A - (cboswell)
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
= Carl, To follow up on this, you can view protocol and suggestions here: I suspect that UV, more than the dye itself, is toxic to cells and to DNA. In addition to DNA dyes with excitation in the visible range, you could, if possible, use a Histone-GFP chromatin label. The Histone-GFP system has been used successfully in several systems for this purpose, and the fusion protein by itself must have very low toxicity, if any, as transformed fly lines carrying it have been around for years now. I have followed Drosophila embryos through several nuclear/cell division cycles with this system, and those embryos can keep on developing. In any event, it would be good to have a biological test (cell viability, cell division, continuation of development) to make sure your imaging regime is not having adverse effects on your specific question. Julio. On Mar 12, 2008, at 12:23 PM, Carl Boswell wrote:
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Carl and group,
I have been using a stable GFP-H2B-HeLa cell line that is easily maintained for live cell imaging. Transient transfection with a GFP-Histone construct will be more work, but for longer term timelapse, like Julio suggested, it may be very effective. The images are fabulous with respect to nuclear/chromatin morphology. I have time lapse imaged with widefield illumination over 48 hours and the cells divide like mad. You add Hoechst to the same cells for time lapse and they die out within 4-8 hours (fabulous images of apoptosis though with the GFP-H2B). Hoechst 33348 or 33258 with UV excitation is really limiting for time lapse imaging and I have moved away from that completely. Molecular Probes has a nuclear envelope marker for live cell imaging I believe, but this may not suit your purposes. Cheers Farid On Wed, Mar 12, 2008 at 6:24 PM, Julio Vazquez <[hidden email]> wrote:
-- Farid Jalali MSc Senior Research Technician/ Lab Manager Applied Molecular Oncology Princess Margaret Hospital Toronto, Canada 416-946-4501 X4351 (Princess Margaret Hospital) 416-581-7754 STTARR at MaRS Building 416-581-7791 STTARR Microscopy Suite |
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In reply to this post by Mario-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I've used DAPI on live cells, and they went through mitosis ok, but these were just in the short term (a couple of hours). I've not managed to get either DAPI or Hoescht to work so well on longer term cultures - probably as everyone says because of the UV issue. Michelle On 12/3/08 22:09, "Mario Moronne" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Carl, Charles, > > The response Charles described is to be expected using Hoechst, if UV > (=< 405 nm) excitation is used. Same goes for DAPI, with the > additional problem that DAPI requires permeabilized cells making it > unsuitable for most live cell experiments. > > I have not tried this but I am sure someone out there probably has, > namely, multi-photon confocal either 2 P or 3 P to excite Hoechst, > which is PM permeable. UV by itself will elicit radical generation > and strand breaking. Hoechst being a minor groove DNA dye could lead > to a lower risk of scission when using 2 P or 3 P to get excitation, > and might pose less risk of collateral DNA damage including unwanted > crosslinking. > > Further, I would be interested to know whether Hoechst added to cells > in culture with no light excitation and maintained through what would > be a couple of passages would permit any cell division. I.E., does > the dye+light abolish cell division or dye by itself? > > Regards All, > Mario > > PS I know that MP illumination can really cook cells, too. Guys what > about using DRAQ5 to watch mitosis? > > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Dear all, >> What is the consensus on using either DAPI or Hoechst staining on >> live cells? Are there effects on mitosis and/or chromosome movement? >> Thanks, >> >> Carl >> >> Carl A. Boswell, Ph.D. >> Molecular and Cellular Biology >> University of Arizona >> 520-954-7053 >> FAX 520-621-3709 > |
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In reply to this post by Boswell, Carl A - (cboswell)
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Carl Boswell <[hidden email]> writes: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear all, > What is the consensus on using either DAPI or Hoechst staining on live > cells? Are there effects on mitosis and/or chromosome movement? What about using a fluorescent protein histone? I have seen nice movies of mitosis using these. Ian |
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In reply to this post by Boswell, Carl A - (cboswell)
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Carl, I have used Hoechst 33342 for some time, because I had primary cells that I could not transfect. I checked the hoechst effect on Hela cells to see what I could expect. The used Hoechst concentration is very improtant. Above 5 µg/ml I killed the cells already in the incubator. With 1 µg/ml I could keep the cells alive in the incubator for days. I used Hela cells with H2B-GFP in it and compared the effect of Hoechst with a 488 laser (to excite GFP) and 800nm multi-photon laser to excite Hoechst. I did 14h experiments. Addition of Hoechst from 0.1 to 5 µg/ml gives a dose dependent negative effect on the number of dividing cells, GFP 488 excitation (and positive on the number of apoptotic cells). Using 800 nm to excite Hoechst added an extra negative effect. With 0.5 µg/ml Hoechst the number of dividing cells dropped from 50% (H2B-GFP 488 excitation as my positive control) to 20% in 800 nm Hoechst excitation. With Hoechst dosis higher or equal to 1 µg/ml I hardly saw dividing cells. Recently it became clear that the Hoechst binding make the cells very sensitive to DNA damage. Especially with the 405 laser you easily damage the DNA. With a more sensitive multi-photon system (I have no non-descanned detectors) the system may be somewhat improved. Best regards, Gert van Cappellen on 12-3-2008 20:23 Carl Boswell said the following: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear all, > What is the consensus on using either DAPI or Hoechst staining on live > cells? Are there effects on mitosis and/or chromosome movement? > Thanks, > > Carl > > Carl A. Boswell, Ph.D. > Molecular and Cellular Biology > University of Arizona > 520-954-7053 > FAX 520-621-3709 -- wigGert van Cappellen, [hidden email] !!! NEW TEL +31-10-70 43578; FAX +31-10-7044736 !!! Assistant professor Dept. of Reproduction and Development; http://www.erasmusmc.nl/rede Optical Imaging Centre; http://www.erasmusmc.nl/oic/ Erasmus MC, room Ee914, Dr. Molenwaterplein 50, 3015 GE ROTTERDAM, The Netherlands Delivery adres: Erasmus MC, Westzeedijk 353, 3015 AA Rotterdam, The Netherlands |
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In reply to this post by Boswell, Carl A - (cboswell)
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal My experience agrees with Gert. I've successfully imaged cells with hoechst using 780 nm for 2 photon and still seen cell division. This was in MEFs and HeLa cells. I was also imaging GFP, RFP and a far red dye in the same cells, and could keep them alive with division for 3 days (probably longer, but I didn't try). Obviously the key is to minimise light input. I titred down the amount of hoechst added so that I kept cells alive (same division rate as controls), and added the minimum of light to the system. At the dose I used, I saw no effect on cell viability either with or without 780nm excitation. In my experience, there is absolutely no way of performing live cell imaging with UV excitation; if you want blue dyes you need to use 2 photon. UV causes too much cellular damage, even if you keep the input low enough so that the cells survive, you are causing too much cellular damage to trust your data. So I would titre the hoechst to a satisfactory level for your cell line, and use a 2 photon. If you don't have access to one, histone-FPs are a good alternative and move you away from UV excitation (assuming your cells are transfectable). |
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In reply to this post by Michelle Peckham
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I have used Hoechst as a counter stain in an eGFP-Microtubuli cell line for studying Mitosis DAPI worked not well in my cells. I also used uv excitation on a widefield system with subsequent deconvolution (with a confocal setting it does not work) but it was very difficult to find the illumination settings preventing the cells from mitotic arrest (which you will get immediately if you illuminate to much) and still get an acceptable signal to noise ratio. But with these settings I was able to collect mitosis over 48 hours, Hoechst was obviously not very toxic within this time frame. In contrast, DRAQ5 was definitely toxic to living cells. You also can try the Vybrant DyeCycle violet stain (Invitrogen - no commercial interest) which is better excitable at 405nm than Hoechst. In sum, you can try and it works but is not optimal. The best way of course would be, as already mentioned, a H2B-fluorescent protein label. Jo Dipl. Biol. Joachim Hehl Staff Scientist LMC-Light Microscopy Centre, ETH Zurich Hönggerberg Institute for Biochemistry Schafmattstrasse 18, HPM F16.1 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6202 Fax: +41 44 632 1298 e-mail: [hidden email] On 3/13/08 9:49 AM, "Michelle Peckham" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > I've used DAPI on live cells, and they went through mitosis ok, but these > were just in the short term (a couple of hours). I've not managed to get > either DAPI or Hoescht to work so well on longer term cultures - probably as > everyone says because of the UV issue. > > Michelle > > > On 12/3/08 22:09, "Mario Moronne" <[hidden email]> wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Carl, Charles, >> >> The response Charles described is to be expected using Hoechst, if UV >> (=< 405 nm) excitation is used. Same goes for DAPI, with the >> additional problem that DAPI requires permeabilized cells making it >> unsuitable for most live cell experiments. >> >> I have not tried this but I am sure someone out there probably has, >> namely, multi-photon confocal either 2 P or 3 P to excite Hoechst, >> which is PM permeable. UV by itself will elicit radical generation >> and strand breaking. Hoechst being a minor groove DNA dye could lead >> to a lower risk of scission when using 2 P or 3 P to get excitation, >> and might pose less risk of collateral DNA damage including unwanted >> crosslinking. >> >> Further, I would be interested to know whether Hoechst added to cells >> in culture with no light excitation and maintained through what would >> be a couple of passages would permit any cell division. I.E., does >> the dye+light abolish cell division or dye by itself? >> >> Regards All, >> Mario >> >> PS I know that MP illumination can really cook cells, too. Guys what >> about using DRAQ5 to watch mitosis? >> >> >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> Dear all, >>> What is the consensus on using either DAPI or Hoechst staining on >>> live cells? Are there effects on mitosis and/or chromosome movement? >>> Thanks, >>> >>> Carl >>> >>> Carl A. Boswell, Ph.D. >>> Molecular and Cellular Biology >>> University of Arizona >>> 520-954-7053 >>> FAX 520-621-3709 >> |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal In addition to the Vybrant DyeCycle Violet (369/437) mentioned here, there are Green (506/534) and Orange (519/563) DyeCycle variants. Those excitation wave lengths should be better. For blood cells by FLOW, we have seen no negative effects on cell health or proliferation, but imaging on adherent cells is more case by case, since illumination conditions are so much more variable and always more intense. Commercial bias acknowledged.... Mike Ignatius Molecular Probes/Invitrogen -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Joachim Hehl Sent: Thursday, March 13, 2008 7:57 AM To: [hidden email] Subject: Re: DNA dyes Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I have used Hoechst as a counter stain in an eGFP-Microtubuli cell line for studying Mitosis DAPI worked not well in my cells. I also used uv excitation on a widefield system with subsequent deconvolution (with a confocal setting it does not work) but it was very difficult to find the illumination settings preventing the cells from mitotic arrest (which you will get immediately if you illuminate to much) and still get an acceptable signal to noise ratio. But with these settings I was able to collect mitosis over 48 hours, Hoechst was obviously not very toxic within this time frame. In contrast, DRAQ5 was definitely toxic to living cells. You also can try the Vybrant DyeCycle violet stain (Invitrogen - no commercial interest) which is better excitable at 405nm than Hoechst. In sum, you can try and it works but is not optimal. The best way of course would be, as already mentioned, a H2B-fluorescent protein label. Jo Dipl. Biol. Joachim Hehl Staff Scientist LMC-Light Microscopy Centre, ETH Zurich Hönggerberg Institute for Biochemistry Schafmattstrasse 18, HPM F16.1 CH-8093, Zurich, Switzerland Web: www.lmc.ethz.ch Phone: +41 44 633 6202 Fax: +41 44 632 1298 e-mail: [hidden email] On 3/13/08 9:49 AM, "Michelle Peckham" <[hidden email]> wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > I've used DAPI on live cells, and they went through mitosis ok, but these > were just in the short term (a couple of hours). I've not managed to get > either DAPI or Hoescht to work so well on longer term cultures - probably as > everyone says because of the UV issue. > > Michelle > > > On 12/3/08 22:09, "Mario Moronne" <[hidden email]> wrote: > >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Carl, Charles, >> >> The response Charles described is to be expected using Hoechst, if UV >> (=< 405 nm) excitation is used. Same goes for DAPI, with the >> additional problem that DAPI requires permeabilized cells making it >> unsuitable for most live cell experiments. >> >> I have not tried this but I am sure someone out there probably has, >> namely, multi-photon confocal either 2 P or 3 P to excite Hoechst, >> which is PM permeable. UV by itself will elicit radical generation >> and strand breaking. Hoechst being a minor groove DNA dye could lead >> to a lower risk of scission when using 2 P or 3 P to get excitation, >> and might pose less risk of collateral DNA damage including unwanted >> crosslinking. >> >> Further, I would be interested to know whether Hoechst added to cells >> in culture with no light excitation and maintained through what would >> be a couple of passages would permit any cell division. I.E., does >> the dye+light abolish cell division or dye by itself? >> >> Regards All, >> Mario >> >> PS I know that MP illumination can really cook cells, too. Guys what >> about using DRAQ5 to watch mitosis? >> >> >>> Search the CONFOCAL archive at >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> Dear all, >>> What is the consensus on using either DAPI or Hoechst staining on >>> live cells? Are there effects on mitosis and/or chromosome movement? >>> Thanks, >>> >>> Carl >>> >>> Carl A. Boswell, Ph.D. >>> Molecular and Cellular Biology >>> University of Arizona >>> 520-954-7053 >>> FAX 520-621-3709 >> |
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In reply to this post by Gert van Cappellen
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal There was a thread a few years ago regarding the inhibition of cell division by Syto dyes, which are excited at 488-514 nm. Aside from issues of phototoxicity and ROS generation, you have many molecules binding to the chromosome which may interfere with transcription of factors/proteins needed for metabolic maintenance and preparation for mitosis. 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 Mar 13, 2008, at 6:13 AM, Gert van Cappellen wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Dear Carl, > > I have used Hoechst 33342 for some time, because I had primary > cells that I could not transfect. I checked the hoechst effect on > Hela cells to see what I could expect. The used Hoechst > concentration is very improtant. Above 5 µg/ml I killed the cells > already in the incubator. With 1 µg/ml I could keep the cells alive > in the incubator for days. I used Hela cells with H2B-GFP in it and > compared the effect of Hoechst with a 488 laser (to excite GFP) and > 800nm multi-photon laser to excite Hoechst. I did 14h experiments. > > Addition of Hoechst from 0.1 to 5 µg/ml gives a dose dependent > negative effect on the number of dividing cells, GFP 488 excitation > (and positive on the number of apoptotic cells). Using 800 nm to > excite Hoechst added an extra negative effect. With 0.5 µg/ml > Hoechst the number of dividing cells dropped from 50% (H2B-GFP 488 > excitation as my positive control) to 20% in 800 nm Hoechst > excitation. With Hoechst dosis higher or equal to 1 µg/ml I hardly > saw dividing cells. Recently it became clear that the Hoechst > binding make the cells very sensitive to DNA damage. Especially > with the 405 laser you easily damage the DNA. With a more sensitive > multi-photon system (I have no non-descanned detectors) the system > may be somewhat improved. > > Best regards, > Gert van Cappellen > > on 12-3-2008 20:23 Carl Boswell said the following: >> Search the CONFOCAL archive at >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >> >> Dear all, >> What is the consensus on using either DAPI or Hoechst staining on >> live cells? Are there effects on mitosis and/or chromosome movement? >> Thanks, >> >> Carl >> >> Carl A. Boswell, Ph.D. >> Molecular and Cellular Biology >> University of Arizona >> 520-954-7053 >> FAX 520-621-3709 > > -- > wigGert van Cappellen, [hidden email] > !!! NEW TEL +31-10-70 43578; FAX +31-10-7044736 !!! > Assistant professor > Dept. of Reproduction and Development; http://www.erasmusmc.nl/rede > Optical Imaging Centre; http://www.erasmusmc.nl/oic/ > Erasmus MC, room Ee914, Dr. Molenwaterplein 50, 3015 GE ROTTERDAM, > The Netherlands > Delivery adres: > Erasmus MC, Westzeedijk 353, 3015 AA Rotterdam, The Netherlands |
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