Benchmarks
Influence of cell growth conditions and medium composition on
EGFP photostability in live cells Anastasia V. Mamontova, Alexey M. Bogdanov, and Konstantin A. Lukyanov
Institute of Bioorganic Chemistry, Moscow, Russia
BioTechniques 58:258-261 (May 2015) doi 10.2144/000114289 Keywords: green fluorescent protein; photobleaching; fluorescence microscopy; antioxidants Supplementary material for this article is available at
www.BioTechniques.com/article/114289.
Photostability is a key characteristic of fluorescent proteins. It was recently demonstrated that green fluorescent protein (GFP) photobleaching in live cells can be suppressed by changes in medium composition. Here we show that Ham’s F12 medium provides very high enhanced GFP (EGFP) photostability during fluorescence microscopy of live cells. This property of Ham’s F12 medium is associated with decreased concentrations of riboflavin and pyridoxine, and increased concentrations of FeSO4
, cyanocobalamine, lipoic
acid, hypoxanthine, and thymidine compared with DMEM. We also found that the rate of EGFP photobleaching strongly depends on cell growth conditions such as cell density and the concentration of serum. We conclude that both imaging medium composition and the physiological state of the cells can strongly affect the photostability of fluorescent proteins. Thus, accurate comparison of the photostabilities of fluorescent proteins should be performed only in side-by-side analysis in identical cell growth conditions and media.
Two decades ago, the application of green fluorescent protein (GFP) as a genetically encoded tag opened a new era in fluores- cence labeling of live cells and organisms (1). Due to their unique ability to form a fluorophore autocatalytically, GFP-like proteins are widely used to follow gene expression, label proteins of interest, and monitor cell physiology in real time (2). Wild-type GFP from the jellyfish
Aequorea victoria possesses subop- timal spectra and inefficient folding in the
METHOD SUMMARY
We determined conditions of cell growth and imaging that strongly increase photostability of enhanced green fluorescent protein (EGFP) in fluorescence microscopy of live cells. Growth at high cell confluency with a high serum concentration increases EGFP photostability, while imaging in Ham’s F12 medium ensures very low rate of photobleaching.
Vol. 58 | No. 5 | 2015 258
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cells of higher animals. These problems were solved by mutagenesis shortly after the introduction of GFP. One of the first artificial mutants of GFP was enhanced GFP (EGFP) (3). In spite of more recent developments
in improving green
fluorescent proteins, EGFP remains one of the most commonly used tags due to the availability of a huge collection of EGFP-labeled proteins, stable cell lines, and transgenic organisms. For example, the plasmid repository AddGene offers
more than 2500 vectors with EGFP, and the Jackson Laboratory has more than 400 EGFP-expressing transgenic mouse strains. Along with fluorescence brightness
and maturation efficiency, photostability represents an important characteristic of a fluorescent protein. Applications such as time-lapse microscopy, recon- struction of 3-D images, single-molecule detection, and fluorescence correlation spectroscopy suffer due to insufficient photostability. To improve photostability of fluorescent proteins, site-directed and random mutagenesis have been used extensively (2). As an alternative approach, optimization of imaging medium compo- sition has been suggested recently (4,5). This approach is based on the fact that GFPs can participate in light-driven redox reactions with intracellular electron acceptors that result in oxidative green- to-red photoconversion (6). Correspond- ingly, suppression of these reactions by changes in medium composition can lead to the enhancement of photosta- bility in the green channel. The simplest solution is to use minimal salt media without any redox-active compounds, such as phosphate-buffered saline (PBS) or Hank’s balanced salt solution, to ensure a several-fold increase of EGFP photosta- bility (4). However, such media lack many essential components required for normal cell physiology and are suitable only for short-term cell maintenance. Therefore, minimally depleted media, which contain all the components of complete media except those that directly decrease EGFP photostability, are preferable for fluores- cence microscopy. In particular, media without riboflavin and pyridoxal (both oxidants) and/or containing rutin (an antioxidant) were found to dramatically enhance the photostability of EGFP (4,5). Addition of rutin represents an
especially promising approach, since it is potentially compatible with a wide variety of experimental models; however, we faced some technical problems during experimentation when we tried adding rutin to our media. First, rutin has low solubility in water. Second, rutin
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