Re: DNA dyes

Posted by Gert van Cappellen on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DNA-dyes-tp592122p592124.html

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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]

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