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** vendor reply **
Hi, Simon,
Backdrop reagents are dye-based quenchers in a buffer solution. Beyond that, unfortunately, the identity of the components is proprietary.
We haven't heard of any problems with its use for live cell assays, with the exception, of course, of assays that require a dye in the extracellular media or on the cell surface in the same wavelength (which would contact the quencher dye).
Jason
Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division
T 1 800 955 6288 then option 4, then option 6, or 541 335 0353 . F 541 335 0238
29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
www.invitrogen.com/technicalsupport
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of simon walker
Sent: Friday, September 14, 2012 1:32 AM
To: [log in to unmask]
Subject: Re: Background fluorescence problem
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Thanks for the various responses. Yes, I'd seen the Bogdanov paper and the Evrogen medium and thought that might be worth a try. The problem we have is that for our assay the culture medium is absolutely critical (it's not just a case of keeping cells alive), so we can't use a minimal HEPES-based buffer. I am interested to know what is in the 'BackDrop' solution. We can't use it unless we're fairly confident it's not going to affect our assay.
Simon
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of George McNamara
Sent: 14 September 2012 01:57
To: [log in to unmask]
Subject: Re: Background fluorescence problem
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Hi Simon,
likely riboflavin and possibly other flavins. See http://www.evrogen.com/products/medium_DMEM_gfp/medium_DMEM_gfp.shtml
and the Bogdanov et al paper referenced at the bottom of the page;
* Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov
S, Lukyanov KA. Cell culture medium affects GFP photostability: a
solution. Nat Methods. 2009; 6 (12):859-60. / pmid: 19935837
<http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=19935837&dopt=Abstract>
Their solution: incubate cells in miedia without (or with low, if
needed) riboflavin for a day.
As a bonus, riboflavin quenches (FRET?) and/or transiently photoconverts GFP to red fluorescence (might be mostly dark states):
Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones. </pubmed/20856676>* Matsuda* A, Shao L, Boulanger J, Kervrann C, Carlton PM, Kner P, Agard D, *Sedat* JW. PLoS One. 2010 Sep 15;5(9):e12768. PMID: 20856676
If you contact Essen Biosciences, they will (hopefully) give you a copy of their application note on the concentrations of riboflavin in many culture media and correlation with fluorescence of those media. Speaking of Essen - they finally introduced a dual green+red fluorescence Incucyte.
Enjoy,
George
On 9/13/2012 11:04 AM, Simon Walker wrote:
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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>
> Dear List,
> We are imaging very weakly fluorescent live cells (expressing GFP) on
> a wide- field system and having issues with a source of background fluorescence.
> When we look at our cells under epi-illumination we see a rapid drop
> in a weak background signal (not where the cells are) that fully
> recovers over a ~10 s period after the illumination light is switched
> off. Our experiments require the use of DMEM as the imaging medium
> and this is the likely cause of problem. It appears that something in
> the medium is sticking to the coverglass. It's not phenol red as the
> effect is seen with both phenol red-containing and phenol- red-free
> DMEM. Does anyone know what else it could be? Has anyone else seen
> anything similar? We're wondering if it could be riboflavin which is in the DMEM we're using. Would this stick to glass?
>
> I've seen that Life Technologies now market a substance that allegedly
> surpresses background fluorescence in DMEM:
> http://products.invitrogen.com/ivgn/product/R37603
> Has anyone tried this? Does anyone know how it works?
>
> Thanks,
> Simon
>
>
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