Posted by
Joachim Hehl on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DNA-dyes-tp592122p592129.html
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalI 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
>>