DNA dyes during mitosis

[Tim Feinstein]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi folks, 

This follows up on a few previous discussions regarding which DNA dye works best for imaging mitotic progression.  A firm consensus of listers says to avoid using the 405 nm laser because it causes major phototoxic effects, especially when labeling DNA.  Most green/red/far red dyes block mitotic progression although the vybrant DyeCycle dyes came up as an option: 

http://confocal-microscopy-list.588098.n2.nabble.com/DNA-dyes-tp592122p592130.html

These dyes are marketed for flow cytometry but they seem to work for imaging as well.  They apparently have no effect on the cell cycle and you can get versions excited at 405, 488 and 530 nm.  Cytoskeleton, Inc. offers another dye called SiR-DNA that excites in far red (652 nm) or even farther red (689 nm) that also (according to marketing) does not interfere with mitosis.  No commercial interest for either company.  

Due to co-staining constraints we are hoping to use the DyeCycle 488.  Has anyone used DC488 to follow mitotic progression?  The paper below suggests you can see it with a FV1000, but they did not do a mitotic time course.  

http://www.jbc.org/content/287/38/31703.full

Thanks!

Tim

Timothy Feinstein, Ph.D. 

Research Scientist

University of Pittsburgh Department of Developmental Biology

From: Confocal Microscopy List <[hidden email]> on behalf of Antonio Jose Pereira <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Friday, September 30, 2016 at 10:06 AM
To: "[hidden email]" <[hidden email]>
Subject: PA-GFP

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi,

We're using 405nm laser to activate PA-GFP tubulin.

We're witnessing a behaviour which was unexpected: we see the much-wanted sudden signal increase in the subsequent GFP channel acquisitions (488 excitation) but the issue is that the signal keeps increasing, sometimes to as much as 130%, in a timescale of the order of a few minutes after the 405 pulse.

After this couple of minutes, either the increase is not significant anymore or it is potentially masked by contributions of a decay resulting from residual turnover (although we're doing taxol stabilization) or photo-bleaching.

We essentially ruled out some residual contribution of the 488 imaging laser to photo-conversion (however, this was performed in the absence of an initial 405 pulse).

Any ideas what this might mean?
Is it any meaningful to think of a population of pa-gfp molecules which undergoes a delayed conversion to the right conformation ...?
Has anyone come across this phenomenon of an increase in signal when trying to calculate photobleaching using Taxol-stabilized microtubules?

I appreciate your input.

Antonio

António José Pereira - Chromosome Instability and Dynamics lab
i3S / IBMC , Universidade do Porto, [phone] (ext 6011)