Re: DNA dyes

Posted by Glen MacDonald-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DNA-dyes-tp592122p592126.html

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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
[phone]
[hidden email]

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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 [phone]
>
> --
> wigGert van Cappellen, [hidden email]
> !!! NEW TEL [phone]; FAX [phone] !!!
> Assistant professor
> Dept. of Reproduction and Development; http://www.erasmusmc.nl/rede
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