Re: DyeCycle and other dyes for cell cycle progression **Vendor reply**
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Kilgore, Jason A. |
Re: DyeCycle and other dyes for cell cycle progression **Vendor reply**
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Tim Feinstein |
Re: DyeCycle and other dyes for cell cycle progression **Vendor reply**
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mmodel |
Re: DyeCycle and other dyes for cell cycle progression **Vendor reply**
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Hi Tim, We estimate the cell cycle phase by a combination of cell volume and Hoechst staining in a manner similar to what they do in flow cytometry. A little crude but better than nothing (depending on what you are looking for). If you are interested I can send you the experimental details.
Mike From: Confocal Microscopy List <[hidden email]> on behalf of Feinstein, Timothy N <[hidden email]>
Sent: Tuesday, November 1, 2016 8:36 PM To: [hidden email] Subject: Re: DyeCycle and other dyes for cell cycle progression **Vendor reply** *****
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***** Thanks Jason, I should have emphasized again that imaging the cell cycle is an entirely off-label use of DyeCycle. We were basically grasping at straws for an easy alternative to fluorescent protein expression, which is not always practical. All the best, Tim Timothy Feinstein, Ph.D. Research Scientist University of Pittsburgh Department of Developmental Biology -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Kilgore, Jason A. Sent: Tuesday, November 01, 2016 6:31 PM To: [hidden email] Subject: Re: DyeCycle and other dyes for cell cycle progression **Vendor reply** ***** To join, leave or search the confocal microscopy listserv, go to: https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Ctnf8%40PITT.EDU%7C2779676da0f34732593b08d402a6e069%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=wsVzmkLTBxInvEZr3%2BfN%2BBsDAJiW1UDtoM%2FBpPaHAE8%3D&reserved=0 Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%7C2779676da0f34732593b08d402a6e069%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=bM8STuJccdSwC6kcmm3CGVVABbfS3c1wZaA8%2Bt8cDGw%3D&reserved=0 and include the link in your posting. ***** ** 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 29851 Willow Creek Rd. Eugene, OR 97402-9132 1-800-955-6288 then option 4, then option 6, then option 2. Or dial direct at +1 541 335 0353 [hidden email] https://na01.safelinks.protection.outlook.com/?url=www.lifetechnologies.com&data=01%7C01%7Ctnf8%40PITT.EDU%7C2779676da0f34732593b08d402a6e069%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=zTkQvjRw5I8kpd2X%2BajcyFtKW4cU7gh897iQyDSjVDA%3D&reserved=0 This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information. Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system. -----Original Message----- From: Confocal Microscopy List [[hidden email]] On Behalf Of Feinstein, Timothy N Sent: Friday, October 28, 2016 12:27 PM To: [hidden email] Subject: DyeCycle and other dyes for cell cycle progression ***** To join, leave or search the confocal microscopy listserv, go to: https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Ctnf8%40PITT.EDU%7C2779676da0f34732593b08d402a6e069%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=wsVzmkLTBxInvEZr3%2BfN%2BBsDAJiW1UDtoM%2FBpPaHAE8%3D&reserved=0 Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Ctnf8%40PITT.EDU%7C2779676da0f34732593b08d402a6e069%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1&sdata=bM8STuJccdSwC6kcmm3CGVVABbfS3c1wZaA8%2Bt8cDGw%3D&reserved=0 and include the link in your posting. ***** 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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