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Hi Simon,

Your cells might not need the 100x excess of riboflavin present in 
"standard" DMEM, your background could be reduced. The Essen tech note I 
mentioned lists:
DMEM 0.4 mg/L riboflavin ... 43.6 units fluorescence
Eagles MEM 0.1 mg/mL     ... 12.9
F12K            0.04               ... 5.4
EBM             0.004             ... 3.7
Riboflavin (alone) 0.4          ... 58.7 (perhaps suggesting that 
culture media quenches riboflavin or it gets converted in part to 
something less fluorescent?)
Contact Essen if you want the entire tech note.


If you absolutely require a green fluorescent protein, spend the time to 
switch to the new Clover or "V6" from Steven Vogel (available from 
addgene.org as VVVVVV).
If you do not need green, switch to tdTomato or the new mRuby2.



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Nat Methods. <#> 2012 Sep 9. doi: 10.1038/nmeth.2171. [Epub ahead of print]


  Improving FRET dynamic range with bright green and red fluorescent
  proteins.

Lam AJ 
</pubmed?term=Lam%20AJ%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
St-Pierre F 
</pubmed?term=St-Pierre%20F%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Gong Y 
</pubmed?term=Gong%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Marshall JD 
</pubmed?term=Marshall%20JD%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Cranfill PJ 
</pubmed?term=Cranfill%20PJ%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Baird MA 
</pubmed?term=Baird%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
McKeown MR 
</pubmed?term=McKeown%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Wiedenmann J 
</pubmed?term=Wiedenmann%20J%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Davidson MW 
</pubmed?term=Davidson%20MW%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Schnitzer MJ 
</pubmed?term=Schnitzer%20MJ%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Tsien RY 
</pubmed?term=Tsien%20RY%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>, 
Lin MZ 
</pubmed?term=Lin%20MZ%5BAuthor%5D&cauthor=true&cauthor_uid=22961245>.


      Source

1] Department of Bioengineering, Stanford University, Stanford, 
California, USA. [2] Department of Pediatrics, Stanford University, 
Stanford, California, USA.


      Abstract

A variety of genetically encoded reporters use changes in fluorescence 
resonance energy transfer (FRET) to report on biochemical processes in 
living cells. The standard genetically encoded FRET pair consists of 
CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic 
range, phototoxicity from the CFP excitation light and complex 
photokinetic events such as reversible photobleaching and 
photoconversion. We engineered two fluorescent proteins,* Clover and 
mRuby2*, which are the brightest green and red fluorescent proteins to 
date and have the highest Förster radius of any ratiometric FRET pair 
yet described. Replacement of CFP and YFP with these two proteins in 
reporters of kinase activity, small GTPase activity and transmembrane 
voltage significantly improves photostability, FRET dynamic range and 
emission ratio changes. These improvements enhance detection of 
transient biochemical events such as neuronal action-potential firing 
and RhoA activation in growth cones.

PMID:
    22961245


PLoS One. <#> 2012;7(5):e38209. Epub 2012 May 30.


  Fluorescence polarization and fluctuation analysis monitors subunit
  proximity, stoichiometry, and protein complex hydrodynamics.

Nguyen TA 
</pubmed?term=Nguyen%20TA%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>, Sarkar 
P 
</pubmed?term=Sarkar%20P%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>, 
Veetil JV 
</pubmed?term=Veetil%20JV%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>, Koushik 
SV 
</pubmed?term=Koushik%20SV%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>, 
Vogel SS 
</pubmed?term=Vogel%20SS%5BAuthor%5D&cauthor=true&cauthor_uid=22666486>.


      Source

Section on Cellular Biophotonics, Laboratory of Molecular Physiology, 
National Institute on Alcohol Abuse and Alcoholism, National Institutes 
of Health, Rockville, Maryland, United States of America.


      Abstract

Förster resonance energy transfer (FRET) microscopy is frequently used 
to study protein interactions and conformational changes in living 
cells. The utility of FRET is limited by false positive and negative 
signals. To overcome these limitations we have developed Fluorescence 
Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule 
based method combining time-resolved fluorescence anisotropy (homo-FRET) 
and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 
nm proximity gauge), brightness (a measure of the number of fluorescent 
subunits in a complex), and correlation time (an attribute sensitive to 
the mass and shape of a protein complex) can be simultaneously measured. 
These measurements together rigorously constrain the interpretation of 
FRET signals. Venus based control-constructs were used to validate FPFA. 
The utility of FPFA was demonstrated by measuring in living cells the 
number of subunits in the ?-isoform of Venus-tagged calcium-calmodulin 
dependent protein kinase-II (CaMKII?) holoenzyme. Brightness analysis 
revealed that the holoenzyme has, on average, 11.9 ± 1.2 subunit, but 
values ranged from 10-14 in individual cells. Homo-FRET analysis 
simultaneously detected that catalytic domains were arranged as dimers 
in the dodecameric holoenzyme, and this paired organization was 
confirmed by quantitative hetero-FRET analysis. In freshly prepared cell 
homogenates FPFA detected only 10.2 ± 1.3 subunits in the holoenzyme 
with values ranging from 9-12. Despite the reduction in subunit number, 
catalytic domains were still arranged as pairs in homogenates. Thus, 
FPFA suggests that while the absolute number of subunits in an 
auto-inhibited holoenzyme might vary from cell to cell, the organization 
of catalytic domains into pairs is preserved.

PMID:
    22666486


I am a bit disappointed Vogel's group did not go for V8 (a well known 
drink) or V12 - the latter either as a polypeptide or with inducible 
dimerization domain. V12 since the goal of this paper is to quantify the 
number of subunits in CaMKIIalpha, which turns out to be 12 (+/- a few) 
as described in 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364239/figure/pone-0038209-g004/ 


On 9/14/2012 4:32 AM, simon walker wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:
>    
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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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