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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:

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