Mamontova Anastasia V, Bogdanov Alexey M, Lukyanov Konstantin A
Institute of Bioorganic Chemistry, Moscow, Russia.
Biotechniques. 2015 May 1;58(5):258-61. doi: 10.2144/000114289. eCollection 2015 May.
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.
光稳定性是荧光蛋白的一个关键特性。最近有研究表明，活细胞中绿色荧光蛋白（GFP）的光漂白可通过培养基成分的改变来抑制。在此我们表明，在活细胞荧光显微镜观察期间，Ham's F12培养基能提供非常高的增强型绿色荧光蛋白（EGFP）光稳定性。与杜氏改良 Eagle培养基（DMEM）相比，Ham's F12培养基的这一特性与核黄素和吡哆醇浓度降低以及硫酸亚铁、氰钴胺、硫辛酸、次黄嘌呤和胸腺嘧啶浓度升高有关。我们还发现，EGFP光漂白速率强烈依赖于细胞生长条件，如细胞密度和血清浓度。我们得出结论，成像培养基成分和细胞的生理状态都能强烈影响荧光蛋白的光稳定性。因此，荧光蛋白光稳定性的准确比较应仅在相同细胞生长条件和培养基下进行并排分析时进行。
