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in aqueous solution remains fully active for only 10–20 h. These factors preclude convenient long-term usage of a concen- trated stock solution of rutin. The stability of rutin and related flavo-
noids in different solutions was studied earlier (7–9). It was demonstrated that rutin is a photostable compound that, unlike the similar flavonoid quercetin, does not undergo decarboxylation under the influence of oxygen in the air (10). Under alkaline conditions (pH ≥ 8.3) in alcoholic solutions, rutin is not degraded, due to the presence of carbohydrate residues that stabilize rutin. Acidification of the media, however, leads to rapid hydrolysis of rutin (11). In the present work, we continued our
efforts in characterizing the influence of cell growth conditions and medium compo- sition on EGFP photostability in order to find optimal conditions for EGFP imaging. Human embryonic kidney 293T
(HEK293T) cells were transfected with the vector EGFP-C1 (Clontech, Palo-Alto, CA) and grown under standard condi- tions (see “Materials and methods” in the Supplementary Materials). Live cells were imaged 48 h after transfection with an AF6000 LX fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Before microscopy, cell culture medium was replaced with either fresh DMEM (as a standard) or a different medium under investigation. A normalized bleaching half-time (time to 50% fluorescence decrease) was used as a quantitative characteristic to compare EGFP photo- stability under different conditions. In our numerous experiments on
measuring photostability of EGFP in live HEK293T cells, we noted that even under apparently the same conditions (i.e., imaging medium composition, light wavelength, and light intensity) photo- bleaching curves can be quite different from one experiment
to another. The
difference in bleaching half-times can be as high as 3–4-fold. We therefore decided to check the influence of several param- eters that may vary in different dishes of growing cells: medium pH, cell confluency, and medium serum concentration. First, we tested DMEM pH from 7.0
to 8.5 and observed no significant differ- ences of EGFP photostability in this pH range (data not shown). Then, we studied EGFP photostability at different cell densities. HEK293T cells were subcul-
Vol. 58 | No. 5 | 2015 B A
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
DMEM F12
10-20% 50-60% 90-100% Cell confluency
0 1 2 3 4 5 6 7 8
C
0.0 0.5 1.0 1.5 2.0 2.5 3.0
DMEM DMEM + composition
DMEM +
FeSO4 0.07 mg/l
DMEM +
FeSO4 0.35 mg/l
Figure 1. Comparison of EGFP photostability in live HEK293T cells under different conditions. Normalized bleaching half-times were used as a measure of EGFP photostability. (A) Influence of cell growth conditions. Values were normalized to the corresponding maxima in DMEM and F12 media. (B) Effect of different media and addition of rutin. The value for DMEM was set to 1. (C) Influence of F12 components. Photostability of EGFP was measured in DMEM alone, DMEM supplemented with a mixture of cyanocobalamine, lipoic acid, hypoxanthine, and thymidine (“DMEM + composition”), or DMEM supplemented with FeSO4
. The value for DMEM was set to 1. 259
www.BioTechniques.com Medium Medium + rutin
2% 10% 20% Serum
DMEM RPMI
F12
DMEMgfp
Relative EGFP photostability
Relative EGFP photostability
Relative EGFP photostability
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