Re: DNA dyes
Posted by
Farid Jalali on
URL: http://confocal-microscopy-list.275.s1.nabble.com/DNA-dyes-tp592122p592134.html
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Carl and group,
I have been using a stable GFP-H2B-HeLa cell line that is easily maintained for live cell imaging. Transient transfection with a GFP-Histone construct will be more work, but for longer term timelapse, like Julio suggested, it may be very effective. The images are fabulous with respect to nuclear/chromatin morphology. I have time lapse imaged with widefield illumination over 48 hours and the cells divide like mad. You add Hoechst to the same cells for time lapse and they die out within 4-8 hours (fabulous images of apoptosis though with the GFP-H2B). Hoechst 33348 or 33258 with UV excitation is really limiting for time lapse imaging and I have moved away from that completely. Molecular Probes has a nuclear envelope marker for live cell imaging I believe, but this may not suit your purposes.
Cheers
Farid
On Wed, Mar 12, 2008 at 6:24 PM, Julio Vazquez <
[hidden email]> wrote:
=
Carl,
To follow up on this, you can view protocol and suggestions here:
I suspect that UV, more than the dye itself, is toxic to cells and to DNA. In addition to DNA dyes with excitation in the visible range, you could, if possible, use a Histone-GFP chromatin label. The Histone-GFP system has been used successfully in several systems for this purpose, and the fusion protein by itself must have very low toxicity, if any, as transformed fly lines carrying it have been around for years now. I have followed Drosophila embryos through several nuclear/cell division cycles with this system, and those embryos can keep on developing. In any event, it would be good to have a biological test (cell viability, cell division, continuation of development) to make sure your imaging regime is not having adverse effects on your specific question.
Julio.
On Mar 12, 2008, at 12:23 PM, Carl Boswell wrote:
Search the CONFOCAL archive at
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
--
Farid Jalali MSc
Senior Research Technician/ Lab Manager
Applied Molecular Oncology
Princess Margaret Hospital
Toronto, Canada
416-946-4501 X4351 (Princess Margaret Hospital)
416-581-7754 STTARR at MaRS Building
416-581-7791 STTARR Microscopy Suite