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Anti-Fading Media for Live Cell GFP Imaging.doc

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Anti-Fading Media for Live Cell GFP Imaging

Anti-FadingMediaforLiveCellGFPImaging AlexeyM.Bogdanov,ElenaI.Kudryavtseva,KonstantinA.Lukyanov* Shemyakin-OvchinnikovInstituteofBioorganicChemistry,Moscow,Russia Abstract Photostabilityisoneofthemostimportantcharacteristicofadyeforfluorescencemicroscopy.Recentlywedemonstrated that vitamins present in imaging media dramatically accelerate photobleaching of Enhanced Green Fluorescent Protein (EGFP) andmany othergreen fluorescent andphotoactivatable proteins. Herewetested allvitamins ofcommonly used media(suchasDulbecco’sModifiedEagleMedium,DMEM)one-by-oneandfoundthatonlytwovitamins,riboflavinand pyridoxal, decrease photostability of EGFP. Thus, DMEM without riboflavin and pyridoxal can be used as an imaging medium,whichensureshighphotostabilityofGFPsattheexpenseofminimalbiochemicaldisturbance.Then,wetested some antioxidants and found that a plant flavonoid rutin greatly enhances photostability of EGFP during live cell microscopy.IncompleteDMEM,rutinincreasedEGFPphotostabilityuptothelevelofvitamin-depletedDMEM.Moreover, beingaddedtovitamin-depletedDMEM,rutinwasabletofurthersuppressEGFPphotobleaching.Potentially,newmedium formulationscanbewidelyusedforfluorescencemicroscopyofGFP-expressingcellsandmodelmulticellularorganismsina varietyofimagingapplications,wherephotostabilityrepresentsachallenge. Citation: Bogdanov AM, Kudryavtseva EI, Lukyanov KA (2012) Anti-Fading Media for Live Cell GFP Imaging. PLoS ONE 7(12): e53004. doi:10.1371/ journal.pone.0053004 Editor:KurtI.Anderson,TheBeatsonInstituteforCancerResearch,UnitedKingdom ReceivedOctober10,2012;AcceptedNovember26,2012;PublishedDecember21,2012 Copyright: ? 2012 Bogdanov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThisworkwassupportedbytheDinastyFoundationandRussianFoundationforBasicResearchgrant11-04-01861a.Thefundershadnoro