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Hi folks, 

It seems like no one has experience with DyeCycle Green for imaging cell
cycle progression, so we bought some and tried it out.  It works great as
a DNA dye, but don't bother trying to image the cell cycle.

Raw image quality is excellent.  You have to titrate it well below
recommended concentrations to avoid saturating the detector.
Interestingly at low dosages it labels mitochondrial DNA almost as
efficiently as nuclear DNA, although you can still easily tell the nucleus
apart.  That might be useful for some folks.  It bleaches fairly readily
with repeated illumination.  Cells proceed through the cell cycle fine
with DyeCycle in the buffer, as long as you don't illuminate it.

That last part was a problem.  We turned our 488 laser (standard Nikon A1
laser launch) down to 0.1% in resonant mode with 4x averaging and about 5
z-sections.  Even at a 30 min sampling interval illuminated cells would
quickly stop moving around and contract their mitochondria.  10 mM Trolox
in the buffer prevented bleaching to a great extent but it did not
completely rescue cell viability.  I have to conclude that nuclear dyes
are just not a good mix with confocal illumination, even when you use
homeopathic levels of light, fast scanning, long time intervals and an
antioxidant buffer supplement.

We also had MitoTracker Red and SiR-Tubulin in the experiment but we ruled
those out as a problem.  Cells were quite happy with those two alone and
proceeded through mitosis while being imaged at 5-min intervals.
SiR-Tubulin was a new reagent for us and we were pleased to see
microtubules even at low dosages (< 100 nM was necessary to allow cell
cycle progression).  It worked best if we just left the it in the buffer
without washing it out.

No commercial interest in any of those.  Just thought this could be useful
to others.  

Best,


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology