CFP/YFP (or any 445/514) FRET slides for sale?

[Gary Laevsky]

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Hi All,

Clemson hooked us up with some awesome 4 color slides.

Anybody make, or know where we can buy, some FRET slides?

My Molecular Expressions slide has finally died.

Thanks in advance.

--
Best,

Gary Laevsky, Ph.D.
Director, Confocal Imaging Facility
Nikon Center of Excellence
Co-Founder, North Atlantic Microscopy Society (NAMS)
https://namsmicroscopy.com/
Dept. of Molecular Biology
Washington Rd.
Princeton University
Princeton, New Jersey, 08544-1014
(O) 609 258 5432
(C) 508 507 1310

[obsydion]

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Hi Gary,

Sorry I don't have a lead but I'm also looking for FRET slides for FLIM and Spectral training. Just wanted to bump this topic in case nobody saw it.

Cheers,
K

[Guillermo Marques-2]

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FocalCheck slide 2 (
https://www.thermofisher.com/order/catalog/product/F36913#/F36913) contains
two shades of green that can be partially resolved with standard CFP/YFP
settings and very well with spectral acquisition and linear unmixing. I
have not tested them for FRET or FLIM, but they certainly work for well
spectral training and QA of spectral microscopes.
Guillermo

On Sat, May 22, 2021 at 12:40 AM Kelvin Poon <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Gary,
>
> Sorry I don't have a lead but I'm also looking for FRET slides for FLIM
> and Spectral training. Just wanted to bump this topic in case nobody saw it.
>
> Cheers,
> K
>

--
Guillermo Marqués
Scientific Director
University Imaging Centers
University of Minnesota
Jackson Hall 1-151
321 Church St SE
Minneapolis, MN55455
Office [phone]
Cell [phone]

[Periasamy, Ammasi (ap3t)-2]

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Hello,
For FRET and FLIM we should use live specimens, particularly for FLIM-FRET work. The lifetime will be reduced for fixed samples due to fixative.
The best way to train the students, you should visit www.addgene.org where you can find FRET standards plasmids, Cerulean-Venus with amino acid linkers (C-5aa-V). This may cost about $60.
This is what we use to train the FRET workshop participants in addition to live cells or participants are allowed to bring their own cells to get trained or to collect FLIM-FRET images.
Hope this helps.
Stay Safe.
Best,
Ammasi

Dr. Ammasi Periasamy
Director and founder of WM Keck Center for Cellular Imaging,
Prof. of Biology & Biomed. Eng., University of Virginia,
Physical & Life Sciences Building (PLSB 005)
90 Geldard Dr., Charlottesville, VA 22904, USA.

http://www.kcci.virginia.edu/people/profile/ap3t
Phone: [phone] or 982-4869
Fax: [phone]
E-mail: [hidden email]

Microscopy Course (Fall Semester) -  http://kcci.virginia.edu/research/microscopy-course
20th Annual FLIM and FRET Workshop-March 7-11, 2022.  http://www.kcci.virginia.edu/workshop





-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Kelvin Poon
Sent: Saturday, May 22, 2021 1:40 AM
To: [hidden email]
Subject: Re: CFP/YFP (or any 445/514) FRET slides for sale?

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Hi Gary,

Sorry I don't have a lead but I'm also looking for FRET slides for FLIM and Spectral training. Just wanted to bump this topic in case nobody saw it.

Cheers,
K

[ICIT]

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Dear Gary,
you can prepare a relatively easy FRET-FLIM specimen with any cell line expressing EGFReceptor (HeLa, CHO). Simply cool the cells on ice and then label them (still on ice) for 10 minutes with a mixture of Alexa488 and Alexa594 labeled EGFs (at concentration of 100 ng/ml). You can then either wash and fix them directly, or even chase them at few minutes intervals and then fix. Only reagents besides the regular stuff are the two labeled EGFs.

The clue is that EGF triggers oligomerization of EGFReceptor and the two EGFs will get within FRET distance from each other to produce pretty massive FRET which is additonally localized at certain regions of the cells. A spicy bonus is that you can study (or demonstrate) the effect of stoichiometry to the FRET efficiency by mixing the two EGFs at different ratios.

Another sample would be to transfect the cells with GPI-eGFP and some 20-24 hours post transfection, while on ice, incubate the cells with Alexa594-labeled Cholera Toxin subunit ß. Wash with cold buffers, fix and proceed as normally. Makes really nice FRET!

This kind of measurements were part of my Ph.D. thesis entitled 'EGF Receptor and Lipid Microdomains' anno 2002.

Cheers,

Mika

[Gary Laevsky]

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Thanks all for the suggestions!

On Sat, May 22, 2021 at 1:42 PM Mika Ruonala <[hidden email]> wrote:

> *****
> 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.
> *****
>
> Dear Gary,
> you can prepare a relatively easy FRET-FLIM specimen with any cell line
> expressing EGFReceptor (HeLa, CHO). Simply cool the cells on ice and then
> label them (still on ice) for 10 minutes with a mixture of Alexa488 and
> Alexa594 labeled EGFs (at concentration of 100 ng/ml). You can then either
> wash and fix them directly, or even chase them at few minutes intervals and
> then fix. Only reagents besides the regular stuff are the two labeled EGFs.
>
> The clue is that EGF triggers oligomerization of EGFReceptor and the two
> EGFs will get within FRET distance from each other to produce pretty
> massive FRET which is additonally localized at certain regions of the
> cells. A spicy bonus is that you can study (or demonstrate) the effect of
> stoichiometry to the FRET efficiency by mixing the two EGFs at different
> ratios.
>
> Another sample would be to transfect the cells with GPI-eGFP and some
> 20-24 hours post transfection, while on ice, incubate the cells with
> Alexa594-labeled Cholera Toxin subunit ß. Wash with cold buffers, fix and
> proceed as normally. Makes really nice FRET!
>
> This kind of measurements were part of my Ph.D. thesis entitled 'EGF
> Receptor and Lipid Microdomains' anno 2002.
>
> Cheers,
>
> Mika
>

--
Best,

Gary Laevsky, Ph.D.
Director, Confocal Imaging Facility
Nikon Center of Excellence
Co-Founder, North Atlantic Microscopy Society (NAMS)
https://namsmicroscopy.com/
Dept. of Molecular Biology
Washington Rd.
Princeton University
Princeton, New Jersey, 08544-1014
(O) 609 258 5432
(C) 508 507 1310