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resulted in a 2-fold photostability increase (Figure 1C). A 5-fold excess of this compound compared with the basic concentration present in Ham’s F12 did not lead to a further increase in photostability (Figure 1C). Interest- ingly, we detected no influence of FeSO4

the photostability of purified EGFP immobi- lized on metal affinity resin (data not shown). Thus, the effect of FeSO4

is a consequence

of some process in live cells, rather than direct interaction with EGFP. The same behavior has been noticed earlier for rutin (5). This work shows that not only imaging

medium composition but also cell growth conditions are important determinants of the rate of EGFP photobleaching. Earlier reports indicate that cells under conditions of low serum or low cell density experience a considerable oxidative shift and an increase in endogenous reactive oxygen species (13). It is reasonable to suggest that the oxidative environment is the cause of decreased EGFP photostability observed in our experiments under these conditions (see Figure 1A). In general, our results suggest that the

physiological state of the live cells can have a strong impact on photostability of fluorescent proteins. This represents an additional signif- icant source of variability for values obtained at different times or in different laboratories. Therefore, we believe that the comparison of photostabilities of fluorescent proteins should be performed only side-by-side under condi- tions as identical as possible; comparison of values from different published reports may be very inaccurate.

Author contributions

A.M.B. and K.A.L. conceived and designed the experiments. A.V.M. and A.M.B. performed the experiments. A.V.M., A.M.B., and K.A.L. analyzed the data and wrote the paper.

Acknowledgments

This work was supported by the Dynasty Foundation and the Molecular and Cell Biology Program of Russian Academy of Sciences. The work was partially carried out using equipment provided by the IBCH Core Facility (CKP IBCH).

Competing interests

A.M.B. and K.A.L. are the inventors on a patent application covering media for GFP imaging.

Vol. 58 | No. 5 | 2015

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2/23/15 7:09 AM shown). In contrast, FeSO4 added to DMEM References

1. Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward, and D.C. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802- 805.

on

2. Chudakov, D.M., M.V. Matz, S. Lukyanov, and K.A. Lukyanov. 2010. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev. 90:1103-1163.

3. Cormack, B.P., R.H. Valdivia, and S. Falkow. 1996. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38.

4. Bogdanov, A.M., E.A. Bogdanova, D.M. Chudakov, T.V. Gorodnicheva, S. Lukyanov, and K.A. Lukyanov. 2009. Cell culture medium affects GFP photostability: a solution. Nat. Methods 6:859-860.

5. Bogdanov, A.M., E.I. Kudryavtseva, and K.A. Lukyanov. 2012. Anti-fading media for live cell GFP imaging. PLoS ONE 7:e53004.

6. Bogdanov, A.M., A.S. Mishin, I.V. Yampolsky, V.V. Belousov, D.M. Chudakov, F.V. Subach, V.V. Verkhusha, S. Lukyanov, and K.A. Lukyanov. 2009. Green fluorescent proteins are light-induced electron donors. Nat. Chem. Biol. 5:459-461.

7. Montaña, M.P. and W.A. Massad. 2010. Stability of flavonoids in the presence of riboflavin-photo- generated reactive oxygen species: a kinetic and mechanistic study on quercetin, morin and rutin. Photochem. Photobiol. 86:827-834.

8. Huvaere, K. and B. Sinnaeve. 2012. Flavonoid deactivation of excited state flavins: reaction

monitoring by mass spectrometry. J. Agric. Food Chem. 60:9261-9272.

9. Perron, N.R. and J.L. Brumaghim. 2009. A review of the antioxidant mechanisms of polyphenol compounds related to iron binding. Cell Biochem. Biophys. 53:75-100.

10. Zenkevich, I.G. and A.Y. Eshchenko. 2007. Identi- fication of the products of oxidation of quercetin by air oxygen at ambient temperature. Molecules 12:654-672.

11. Dechene, E.B. 1951. The relative stability of rutin and quercetin in alkaline solution. J Am Pharm Assoc Am Pharm Assoc. 40:495-497.

12. Crossley, R.A., D.J. Gaskin, K. Holmes, F. Mulholland, J.M. Wells, D.J. Kelly, A.H. van Vliet, and N.J. Walton. 2007. Riboflavin biosynthesis is associated with assimilatory ferric reduction and iron acquisition by Campylobacter jejuni. Appl. Environ. Microbiol. 73:7819-7825.

13. Gutscher, M., A.L. Pauleaum, L. Marty, T. Brach, G.H. Wabnitz, Y. Samstag, A.J. Meyer, and T.P. Dick. 2008. Real-time imaging of the intracellular glutathione redox potential. Nat. Methods 5:553-559.

Received 09 January 2015; accepted 09 February 2015.

Address correspondence to Konstantin A. Lukyanov, Institute of Bioorganic Chemistry, Moscow, Russia. E-mail: kluk@ibch.ru

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