Dear Yevgeniy,
While i haven't had any experience imobilising cells for FRET we have had great success in the past imobolising cells for ICS. We had a cell line that expressed the receptor of interest but was round and didn't attache to anything. We just put them in some 1% low melt temp agarose (made up in PBS) and everything worked really well. Now we were only looking at the bleching of one receptor, but any movement in the system would have been noticed in the final analysis and there was none. I cant see a low percentage of agarose interfereing with you FRET measurments.
Cheers
Cam
Cameron J. Nowell
Microscpy Manager
Central Resource for Advanced Microscopy
Ludwig Insttue for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
http://www.ludwig.edu.au/branch/research/platform/microscopy.htm
________________________________
From: Confocal Microscopy List on behalf of Yevgeniy Romin
Sent: Sun 18/04/2010 1:49 AM
To: [log in to unmask]
Subject: Re: Mounting media for FRET experiments
Thank you very much to everyone replying both on and off the list. Your advice and the papers that were referred to were very helpful. FRET on live samples would be the ideal choice, unfortunately in this case we are doing FRET on white blood cells, which are very round and refuse to attach and sit still, introducing motion artifacts into the FRET analysis. We are currently working on immobilizing them, but there is a fear of matrigel or a more viscous media introducing FRET measurement artifacts as well. Does anyone have experience with doing FRET on cells that do not attach or immobilizing them in a least invasive way?
________________________________________
From: Confocal Microscopy List [[log in to unmask]] On Behalf Of Daniel Gitler [[log in to unmask]]
Sent: Saturday, April 17, 2010 5:22 AM
To: [log in to unmask]
Subject: Re: Mounting media for FRET experiments
Dear John and Yevgeniy,
My experience when measuring FRET from the same samples before and after fixation is that fixation itself will very much change the FRET efficiency value that you measure (Cerulean and Venus in HEK cells, PBS for both). Efficiency in the fixed samples was significantly lower. This should not come as any big surprise, since the cellular environment around the fluorophores will be very different.
The possible reasons for the changes in FRET efficiency are very many...
I also do most of my FRET imaging in live samples.
Daniel
----- Original Message -----
From: Vitaly Boyko <[log in to unmask]>
Date: Friday, April 16, 2010 19:36
Subject: Re: Mounting media for FRET experiments
To: [log in to unmask]
> Dear John,
> Dear Yevgeniy,
>
> Yes, PBS or specialized low serum (1-2%) media is broadly used
> for live cell imaging under TIR illumination.
>
> I would like to bring your attention to two papers which are
> relevant to single molecule fluorscence and/or low light live
> cell imaging - I sent them separately to your e-mail addressess
> as the LISTSERVER rejected my message due to the attachments. I
> can also send the papers to anyone by request.
>
> 1. The Use of Protocatechuate Dioxygenase for Maintaining
> Anaerobic Conditions in Biochemical ExperimentsPravin V. Patil
> and David P. BallouDepartment of Biological Chemistry,
> University of Michigan, Ann Arbor, Michigan 48109-0606
> Analytical Biochemistry 286, 187-192 (2000)
> 2. An Oxygen Scavenging System for Improvement of Dye Stability
> in Single-Molecule Fluorescence ExperimentsColin EcheverriŽa
> Aitken,* R. Andrew Marshall,y and Joseph D. Puglisiz§
> Biophysical Journal Volume 94 March 2008 1826-1835
>
> If you have any questions, please contact me offline or phone.
>
> Also, the accuracy of the FRET eff. measurements could vary or
> be affected by the mounting media used - live cell FRET seems to
> be the best approach.
>
> Have a nice weekend,
>
> Vitaly
> 301-515-7833
>
>
>
>
>
> ________________________________
> From: John Oreopoulos <[log in to unmask]>
> To: [log in to unmask]
> Sent: Fri, April 16, 2010 12:01:32 PM
> Subject: Re: Mounting media for FRET experiments
>
> Just regular PBS solution. I have never tried a rigorous
> comparison of different media.
>
> John
>
> On 2010-04-16, at 11:54 AM, Yevgeniy Romin wrote:
>
> > Thank you very much John, I found the paper you recommended
> and it is already proving useful. A question for you then -
> which media were you using for the live cells? What I mean is
> whether it was something like PBS or some specific media?
> >
> > ---------------------------------------------------
> > Yevgeniy Romin
> >
> > Digital Microscopist
> > Memorial Sloan-Kettering Cancer Center
> > Molecular Cytology Core Facility
> > 1275 York Ave. Box 333
> > New York, NY 10065
> > Tel.646-888-2186
> > Fax. 646-422-0640
> > ---------------------------------------------------
> >
> > -----Original Message-----
> > From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf Of John Oreopoulos
> > Sent: Friday, April 16, 2010 11:30 AM
> > To: [log in to unmask]
> > Subject: Re: Mounting media for FRET experiments
> >
> > I have not used fixed specimens for FRET imaging (I have used
> the sensitized emission method on living cells), but I came
> across this paper a while ago which might be of interest to you:
> >
> > Rodighiero, S., et al., Fixation, mounting and sealing with
> nail polish of cell specimens lead to incorrect FRET
> measurements using acceptor photobleaching. Cellular Physiology
> and Biochemistry, 2008. 21(5-6): p. 489-498.
> >
> > John Oreopoulos
> >
> >
> > On 2010-04-16, at 11:20 AM, Yevgeniy Romin wrote:
> >
> >> Dear list
> >>
> >> During a recent FRET experiment a question came up about what
> mounting media should be used on fixed samples during FRET
> experiments and whether any one media is better than other for
> this particular type of experiment. From what's published it
> seems that people use all kinds of different media. Does
> anybody here have any idea about any specific media that would
> be best/give least interference with FRET results?
> >>
> >> Thanks a lot to all of you in advance,
> >>
> >> ---------------------------------------------------
> >> Yevgeniy Romin
> >>
> >> Digital Microscopist
> >> Memorial Sloan-Kettering Cancer Center
> >> Molecular Cytology Core Facility
> >> 1275 York Ave. Box 333
> >> New York, NY 10065
> >> Tel.646-888-2186
> >> Fax. 646-422-0640
> >> ---------------------------------------------------
> >>
> >>
> >>
> >>
> =====================================================================>>
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>
>
Daniel Gitler, Ph.D.
Department of Physiology and Neurobiology
Faculty of Health Sciences
Ben Gurion University of the Negev
Beer-Sheva 84105
Israel
Tel: +972-8-6477345
Cell: +972-54-2110100
Fax: + 972-8-6477628
http://web2.bgu.ac.il/physiology/faculty-members/daniel-gitler/
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