Labeling sperm
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I have a need to label the nucleus of some marine invertebrate sperm to track the movement of the male pronucleus within the egg. Does anyone have a suggestion of a marker that could be incorporated into live sperm without interfering with their ability to fertilize the eggs? Thanks for you help and wisdom. John |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hoechst 33342? I believe I remember a paper on Bryozoan fertilization using that. Julian On 2/17/12 5:05 PM, Pilger, John wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > I have a need to label the nucleus of some marine invertebrate sperm to > track the movement of the male pronucleus within the egg. Does anyone > have a suggestion of a marker that could be incorporated into live sperm > without interfering with their ability to fertilize the eggs? > > Thanks for you help and wisdom. > > John > -- Julian P.S. Smith III Director, Winthrop Microscopy Facility Dept. of Biology Winthrop University 520 Cherry Rd. Rock Hill, SC 29733 803-323-2111 x6427 (vox) 803-323-3448 (fax) 803-524-2347 (cell) Research Website www.birdnest.org/smithj Personal Website www.rociada-east.net |
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In reply to this post by Pilger, John
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi John, Draq5 works well for nuclear staining of live samples (from Biostatus, no commercial interest). We don't have any experience with your kind of experiment, as with any intercalating dye you'll need loads of controls to make sure it doesn't mess up your DNA, but at least you won't need UV excitation (as with most other nuclear staining) - nice red light (~633nm) - so at least the light doesn't do any harm. Good luck, Martin On Fri, 17 Feb 2012 17:05:08 -0500, Pilger, John <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >I have a need to label the nucleus of some marine invertebrate sperm to >track the movement of the male pronucleus within the egg. Does anyone >have a suggestion of a marker that could be incorporated into live sperm >without interfering with their ability to fertilize the eggs? > >Thanks for you help and wisdom. > >John |
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In reply to this post by Pilger, John
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Could a good polarization microscope achieve this? I never tried, but remember that some in vitro fertilization setups use polarization light microscopy (Aberio? - po commercial interest). Stan Vitha On Mon, 20 Feb 2012 12:37:45 -0600, Martin Spitaler <[hidden email]> wrote: >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >Hi John, > > Draq5 works well for nuclear staining of live samples (from Biostatus, no >commercial interest). We don't have any experience with your kind of >experiment, as with any intercalating dye you'll need loads of controls to >make sure it doesn't mess up your DNA, but at least you won't need UV >excitation (as with most other nuclear staining) - nice red light (~633nm) - >so at least the light doesn't do any harm. > >Good luck, > >Martin > > >On Fri, 17 Feb 2012 17:05:08 -0500, Pilger, John <[hidden email]> > >>***** >>To join, leave or search the confocal microscopy listserv, go to: >>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>***** >> >>I have a need to label the nucleus of some marine invertebrate sperm to >>track the movement of the male pronucleus within the egg. Does anyone >>have a suggestion of a marker that could be incorporated into live sperm >>without interfering with their ability to fertilize the eggs? >> >>Thanks for you help and wisdom. >> >>John |
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In reply to this post by Martin Spitaler
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi John, Biostatus doesn't recommend using Draq5 to flow-sort viable cells as it is "ultimately cytotoxic" (quote from package insert). A lot is going to depend on how long you have to follow the stained DNA. For an interesting comparison of live-cell permeant DNA stains in terms of induction of DNA damage, look at Zhao et al, Cytometry A 75A: 510 (2009). They compared Hoechst, Draq5, DyeCycle Violet, and Syto17 and found the least damage in Syto17-stained cells; prolonged exposure to stain generally enhanced DNA damage. There wasn't any imaging involved, so no contribution to damage from extended laser exposure. I have successfully sorted CHO cells stained with DyeCycle Violet (as others have done with Hoechst) and gotten them to grow afterwards. There is also a technique for flow-sorting Hoechst stained sperm (again, minimal laser exposure) to select X- or Y- bearing sperm for generating offspring of the desired sex - as far as I know, just in agricultural species. The brave new world approaches! Regards, Carol On Feb 20, 2012, at 1:37 PM, Martin Spitaler wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi John, > > Draq5 works well for nuclear staining of live samples (from > Biostatus, no > commercial interest). We don't have any experience with your kind of > experiment, as with any intercalating dye you'll need loads of > controls to > make sure it doesn't mess up your DNA, but at least you won't need UV > excitation (as with most other nuclear staining) - nice red light > (~633nm) - > so at least the light doesn't do any harm. > > Good luck, > > Martin > > > On Fri, 17 Feb 2012 17:05:08 -0500, Pilger, John > <[hidden email]> wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> I have a need to label the nucleus of some marine invertebrate >> sperm to >> track the movement of the male pronucleus within the egg. Does anyone >> have a suggestion of a marker that could be incorporated into live >> sperm >> without interfering with their ability to fertilize the eggs? >> >> Thanks for you help and wisdom. >> >> John Carol E. Norris, Ph.D Facility Scientist Flow Cytometry/Confocal Microscopy Facility Biotechnology/Bioservices Center University of Connecticut Unit 3149 91 N. Eagleville Rd Storrs, CT 06269-3149 Phone (860) 486-3080 Fax (860) 486-5005 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Agreed - Draq5 does not kill cells right away but in my experience they won't divide (or, I assume, undergo meiosis) and will eventually die. I have searched for a chemical label to observe DNA during division, and as of 2005 or so that approach seemed like a dead end. IMO for this study you really want to use a genetically encoded histone-GFP. I understand that the process of collecting sperm from wild-caught sea urchins does not give a lot of opportunities for transfection, but it may be the least frustrating option. You could also look up the work of Conly Rieder. He has done amazing work with DNA imaging through the cell cycle with transmitted light using DIC. cheers, TF Timothy Feinstein, PhD Postdoctoral Fellow Laboratory for GPCR Biology Dept. of Pharmacology & Chemical Biology University of Pittsburgh, School of Medicine BST W1301, 200 Lothrop St. Pittsburgh, PA 15261 On Feb 22, 2012, at 9:00 AM, Carol Norris wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi John, > > Biostatus doesn't recommend using Draq5 to flow-sort viable cells as it is "ultimately cytotoxic" (quote from package insert). A lot is going to depend on how long you have to follow the stained DNA. For an interesting comparison of live-cell permeant DNA stains in terms of induction of DNA damage, look at Zhao et al, Cytometry A 75A: 510 (2009). They compared Hoechst, Draq5, DyeCycle Violet, and Syto17 and found the least damage in Syto17-stained cells; prolonged exposure to stain generally enhanced DNA damage. There wasn't any imaging involved, so no contribution to damage from extended laser exposure. I have successfully sorted CHO cells stained with DyeCycle Violet (as others have done with Hoechst) and gotten them to grow afterwards. There is also a technique for flow-sorting Hoechst stained sperm (again, minimal laser exposure) to select X- or Y-bearing sperm for generating offspring of the desired sex - as far as I know, just in agricultural species. The brave new world approaches! > > Regards, > Carol > > > On Feb 20, 2012, at 1:37 PM, Martin Spitaler wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi John, >> >> Draq5 works well for nuclear staining of live samples (from Biostatus, no >> commercial interest). We don't have any experience with your kind of >> experiment, as with any intercalating dye you'll need loads of controls to >> make sure it doesn't mess up your DNA, but at least you won't need UV >> excitation (as with most other nuclear staining) - nice red light (~633nm) - >> so at least the light doesn't do any harm. >> >> Good luck, >> >> Martin >> >> >> On Fri, 17 Feb 2012 17:05:08 -0500, Pilger, John <[hidden email]> wrote: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> I have a need to label the nucleus of some marine invertebrate sperm to >>> track the movement of the male pronucleus within the egg. Does anyone >>> have a suggestion of a marker that could be incorporated into live sperm >>> without interfering with their ability to fertilize the eggs? >>> >>> Thanks for you help and wisdom. >>> >>> John > > > > Carol E. Norris, Ph.D > Facility Scientist > Flow Cytometry/Confocal Microscopy Facility > Biotechnology/Bioservices Center > University of Connecticut Unit 3149 > 91 N. Eagleville Rd > Storrs, CT 06269-3149 > > Phone (860) 486-3080 > Fax (860) 486-5005 |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hoechst is used for sex selection of bull sperm by FACS which is then used for artificial insemination (AI). I believe it has also been used for other species and so may be worth exploring. Concentration may prove to be the vital factor. Labelled bull sperm for AI was is easily seen with the 405 nm laser by confocal microscopy and standard fluorescence microscopy. Best regards Peter On 22/02/2012 14:51, Tim Feinstein wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Agreed - Draq5 does not kill cells right away but in my experience they won't divide (or, I assume, undergo meiosis) and will eventually die. I have searched for a chemical label to observe DNA during division, and as of 2005 or so that approach seemed like a dead end. > > IMO for this study you really want to use a genetically encoded histone-GFP. I understand that the process of collecting sperm from wild-caught sea urchins does not give a lot of opportunities for transfection, but it may be the least frustrating option. You could also look up the work of Conly Rieder. He has done amazing work with DNA imaging through the cell cycle with transmitted light using DIC. > > cheers, > > > TF > > Timothy Feinstein, PhD > Postdoctoral Fellow > Laboratory for GPCR Biology > Dept. of Pharmacology& Chemical Biology > University of Pittsburgh, School of Medicine > BST W1301, 200 Lothrop St. > Pittsburgh, PA 15261 > > On Feb 22, 2012, at 9:00 AM, Carol Norris wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hi John, >> >> Biostatus doesn't recommend using Draq5 to flow-sort viable cells as it is "ultimately cytotoxic" (quote from package insert). A lot is going to depend on how long you have to follow the stained DNA. For an interesting comparison of live-cell permeant DNA stains in terms of induction of DNA damage, look at Zhao et al, Cytometry A 75A: 510 (2009). They compared Hoechst, Draq5, DyeCycle Violet, and Syto17 and found the least damage in Syto17-stained cells; prolonged exposure to stain generally enhanced DNA damage. There wasn't any imaging involved, so no contribution to damage from extended laser exposure. I have successfully sorted CHO cells stained with DyeCycle Violet (as others have done with Hoechst) and gotten them to grow afterwards. There is also a technique for flow-sorting Hoechst stained sperm (again, minimal laser exposure) to select X- or Y-bearing sperm for generating offspring of the desired sex - as far as I know, just in agricultural species. The brave new world approaches! >> >> Regards, >> Carol >> >> >> On Feb 20, 2012, at 1:37 PM, Martin Spitaler wrote: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Hi John, >>> >>> Draq5 works well for nuclear staining of live samples (from Biostatus, no >>> commercial interest). We don't have any experience with your kind of >>> experiment, as with any intercalating dye you'll need loads of controls to >>> make sure it doesn't mess up your DNA, but at least you won't need UV >>> excitation (as with most other nuclear staining) - nice red light (~633nm) - >>> so at least the light doesn't do any harm. >>> >>> Good luck, >>> >>> Martin >>> >>> >>> On Fri, 17 Feb 2012 17:05:08 -0500, Pilger, John<[hidden email]> wrote: >>> >>>> ***** >>>> To join, leave or search the confocal microscopy listserv, go to: >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>>> ***** >>>> >>>> I have a need to label the nucleus of some marine invertebrate sperm to >>>> track the movement of the male pronucleus within the egg. Does anyone >>>> have a suggestion of a marker that could be incorporated into live sperm >>>> without interfering with their ability to fertilize the eggs? >>>> >>>> Thanks for you help and wisdom. >>>> >>>> John >> >> >> Carol E. Norris, Ph.D >> Facility Scientist >> Flow Cytometry/Confocal Microscopy Facility >> Biotechnology/Bioservices Center >> University of Connecticut Unit 3149 >> 91 N. Eagleville Rd >> Storrs, CT 06269-3149 >> >> Phone (860) 486-3080 >> Fax (860) 486-5005 -- Dr Peter O'Toole Head of Imaging and Cytometry Technology Facility Department of Biology (Area 15) University of York YORK YO10 5DD Tel : +44 (0)1904 328722 Fax : +44 (0)1904 328804 email : [hidden email] www.york.ac.uk/biology/tf Times Higher Education University of the Year 2010 EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Good point, Peter. I did not have a 405 laser at the time, and felt a bit conservative at any rate about long-term live imaging with UV excitation, but you are right that Hoechst has been used successfully. Apologies for the memory lapse. cheers, TF Timothy Feinstein, PhD Postdoctoral Fellow Laboratory for GPCR Biology Dept. of Pharmacology & Chemical Biology University of Pittsburgh, School of Medicine BST W1301, 200 Lothrop St. Pittsburgh, PA 15261 On Feb 22, 2012, at 11:31 AM, Peter O'Toole wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hoechst is used for sex selection of bull sperm by FACS which is then used for artificial insemination (AI). I believe it has also been used for other species and so may be worth exploring. Concentration may prove to be the vital factor. Labelled bull sperm for AI was is easily seen with the 405 nm laser by confocal microscopy and standard fluorescence microscopy. > > Best regards > > Peter > > > On 22/02/2012 14:51, Tim Feinstein wrote: >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Agreed - Draq5 does not kill cells right away but in my experience they won't divide (or, I assume, undergo meiosis) and will eventually die. I have searched for a chemical label to observe DNA during division, and as of 2005 or so that approach seemed like a dead end. >> >> IMO for this study you really want to use a genetically encoded histone-GFP. I understand that the process of collecting sperm from wild-caught sea urchins does not give a lot of opportunities for transfection, but it may be the least frustrating option. You could also look up the work of Conly Rieder. He has done amazing work with DNA imaging through the cell cycle with transmitted light using DIC. >> >> cheers, >> >> >> TF >> >> Timothy Feinstein, PhD >> Postdoctoral Fellow >> Laboratory for GPCR Biology >> Dept. of Pharmacology& Chemical Biology >> University of Pittsburgh, School of Medicine >> BST W1301, 200 Lothrop St. >> Pittsburgh, PA 15261 >> >> On Feb 22, 2012, at 9:00 AM, Carol Norris wrote: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> Hi John, >>> >>> Biostatus doesn't recommend using Draq5 to flow-sort viable cells as it is "ultimately cytotoxic" (quote from package insert). A lot is going to depend on how long you have to follow the stained DNA. For an interesting comparison of live-cell permeant DNA stains in terms of induction of DNA damage, look at Zhao et al, Cytometry A 75A: 510 (2009). They compared Hoechst, Draq5, DyeCycle Violet, and Syto17 and found the least damage in Syto17-stained cells; prolonged exposure to stain generally enhanced DNA damage. There wasn't any imaging involved, so no contribution to damage from extended laser exposure. I have successfully sorted CHO cells stained with DyeCycle Violet (as others have done with Hoechst) and gotten them to grow afterwards. There is also a technique for flow-sorting Hoechst stained sperm (again, minimal laser exposure) to select X- or Y-bearing sperm for generating offspring of the desired sex - as far as I know, just in agricultural species. The brave new world approaches! >>> >>> Regards, >>> Carol >>> >>> >>> On Feb 20, 2012, at 1:37 PM, Martin Spitaler wrote: >>> >>>> ***** >>>> To join, leave or search the confocal microscopy listserv, go to: >>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>>> ***** >>>> >>>> Hi John, >>>> >>>> Draq5 works well for nuclear staining of live samples (from Biostatus, no >>>> commercial interest). We don't have any experience with your kind of >>>> experiment, as with any intercalating dye you'll need loads of controls to >>>> make sure it doesn't mess up your DNA, but at least you won't need UV >>>> excitation (as with most other nuclear staining) - nice red light (~633nm) - >>>> so at least the light doesn't do any harm. >>>> >>>> Good luck, >>>> >>>> Martin >>>> >>>> >>>> On Fri, 17 Feb 2012 17:05:08 -0500, Pilger, John<[hidden email]> wrote: >>>> >>>>> ***** >>>>> To join, leave or search the confocal microscopy listserv, go to: >>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>>>> ***** >>>>> >>>>> I have a need to label the nucleus of some marine invertebrate sperm to >>>>> track the movement of the male pronucleus within the egg. Does anyone >>>>> have a suggestion of a marker that could be incorporated into live sperm >>>>> without interfering with their ability to fertilize the eggs? >>>>> >>>>> Thanks for you help and wisdom. >>>>> >>>>> John >>> >>> >>> Carol E. Norris, Ph.D >>> Facility Scientist >>> Flow Cytometry/Confocal Microscopy Facility >>> Biotechnology/Bioservices Center >>> University of Connecticut Unit 3149 >>> 91 N. Eagleville Rd >>> Storrs, CT 06269-3149 >>> >>> Phone (860) 486-3080 >>> Fax (860) 486-5005 > > -- > Dr Peter O'Toole > Head of Imaging and Cytometry > Technology Facility > Department of Biology (Area 15) > University of York > YORK > YO10 5DD > > Tel : +44 (0)1904 328722 > Fax : +44 (0)1904 328804 > email : [hidden email] > www.york.ac.uk/biology/tf > > Times Higher Education University of the Year 2010 > > EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm |
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