tured to achieve 10%–20%, 50%–60%, and 90%–100% confluency by the day of experiment. In DMEM, there was a clear increase of EGFP photostability as cell density increased (Figure 1A). A similar dependence was observed for the cells in F12 medium (see discussion below regarding the use of this medium). Finally, we checked the effect of serum
Fully Automated 3-Color Fluorescence
concentration. Cells were grown in media supplemented with 2%, 10%, or 20% FBS. We found substantial differences in EGFP photostability for both DMEM and F12. The lowest photostability was detected at 2% FBS and the highest at 20%. Differences between the highest and lowest bleaching half-times were 2.5–3-fold for DMEM and 1.3–1.5-fold for F12 (Figure 1A). Earlier, we had shown that a short-term (0.5 h) incubation of cells with or without serum in the imaging medium does not affect EGFP photostability (4). Thus, the long-term effects of serum on cell physiology were important in the present experiments. To minimize the effects of cell physiology
on data reproducibility, further comparative experiments on EGFP photostability were performed on either the same dish or dishes with similar cell densities. Earlier, we had shown that the positive
effect of rutin on EGFP photostability in cells only occurs with freshly prepared water solutions (5). To find appropriate preser- vation conditions, we tested rutin activity after storage in 2 different solvents, water and DMSO, and at different temperatures (22°C, 4°C, -20°C and -70°C). After 3 weeks, rutin in DMSO maintained full activity at each storage temperature. In contrast, the rutin water solution completely lost its activity at 22°C and 4°C; only frozen samples remained active.
Importantly, rutin possesses much higher
solubility in DMSO than in water (approxi- mately 3 g/L and 100 mg/L respectively). Thus, DMSO can be used to prepare a concentrated stock solution and store it for convenience. At the same time, low tempera- tures would be recommended for the storage of an aqueous rutin solution in situations where DMSO cannot be used. Along with DMEM, Ham’s F12 and
Live Cell Imaging
RPMI1640 media are widely used in cell biology. Ham’s F12 was developed for culti- vating rat hepatocytes, epithelial cells of rats, and Chinese Hamster Ovary (CHO) cells. RPMI1640 medium is conventionally used for growing human lymphoid cells but is also suitable for the cultivation of a variety of cell
260
lines. We tested whether rutin has a positive effect on EGFP photostability in these media. All tested media (both with and without
rutin) did not affect the overall initial brightness of EGFP in the cells (Supplementary Figure S1). The photostabilities of HEK293T- expressed EGFP in DMEM and RPMI1640 were almost identical. Addition of rutin to these media caused a similar several-fold increase in EGFP photostability (Figure 1B). In contrast, Ham’s F12 ensured very high EGFP photostability, which was practically unaffected by rutin (Figure 1B). F12 medium demonstrated a very strong
suppression of EGFP photobleaching, so we studied its composition in order to determine the components responsible for this effect. F12 is markedly different in composition compared to DMEM (Supple- mentary Table S1). In particular, F12 contains greatly decreased concentrations of riboflavin and pyridoxine (11- and 67-fold, respec- tively). These differences appear to be most important, as we previously demonstrated that these vitamins reduce EGFP photosta- bility dramatically (4,5). However, we noted that F12 has a
superior effect on EGFP photostability, even compared with DMEMgfp
(DMEM without
vitamins) (Figure 1B). Also, the fact that rutin enhances EGFP photostability in DMEMgfp but not in F12, indicates an ultimate state of EGFP photostability in F12. We next tested the effects of other
substances that are absent or present in much lower concentrations in DMEM. To this end, EGFP photostability in live cells was evaluated in DMEM supplemented with the individual compounds or their combinations, compared to regular DMEM. As expected, we found that alanine, asparagine, aspartate, glutamate, biotin, and putrescine (either alone or all together) have no effect on EGFP photo- stability (data not shown). Potentially redox-active compounds such
as cyanocobalamine (vitamin B12), lipoic acid, hypoxanthine, and thymidine also did not exert any effects on EGFP photobleaching when added to DMEM individually; however, the combination of these compounds provided an approximately 2-fold photosta- bility enhancement (Figure 1C). Metal ions such as Fe2+/3+
and Cu+/2+ are
very reactive, which suggests possible inter- actions with intracellular electron acceptors responsible for the oxidative photoconversion of GFP (12). CuSO4
and ZnSO4 showed no significant effect on photostability (data not
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