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** Vendor reply **
I just wanted to give a bit of input that the DyeCycle dyes are only validated for flow cytometry usage, not for imaging.
For flow, organellar localization and photobleaching are not as much of an issue, as long as there is the appropriate flow pattern is observed for distinguishing the cell cycle stages.
For microscopy and cell cycle, using mammalian cell lines, I would suggest the Premo FUCCI Cell Cycle Sensor, which is fluorescent protein based.
Best,
Jason
Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Life Sciences Solutions
Thermo Fisher Scientific
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Feinstein, Timothy N
Sent: Friday, October 28, 2016 12:27 PM
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Subject: DyeCycle and other dyes for cell cycle progression
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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
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