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Ros in Yeast
Ros in Yeast
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Methods
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This article can be cited before page numbers have been issued, to do this please use: J. James, N. Fiji,
D. Roy, D. Andrew, M. S. Shihabudeen, D. Chattopadhyay and K. Thirumurugan, Anal. Methods, 2015,
DOI: 10.1039/C5AY02278A.
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DOI: 10.1039/C5AY02278A
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16 DOI: 10.1039/x0xx00000x
17 www.rsc.org/
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19 A protocol to efficiently assess Reactive Oxygen Species (ROS)
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could lead to nuances in data interpretation . Several researchers
20 levels in yeast cells using H2DCF-DA is described here. This method use H2DCF-DA to estimate cellular ROS and hence, there exists a
21 employs lithium acetate to permeate the cell wall, and thus, need for more reliable methods. Despite the advent of advanced
22 augments the release of the fluorescent product, techniques such as flow cytometry to analyse fluorescence in intact
23 dichlorofluorescein from the cells. This protocol obviates the need cells, determination of H2DCF-DA oxidation using a fluorescence
for both physical and enzymatic lysis methods that are arduous spectrophotometer is commonplace in several laboratories
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and time consuming. This method is simple, less time consuming worldwide owing to its cost-effectiveness. The usual method for
25 and reproducible, specially while dealing with a large sample size. ROS assessment in yeast involves the incubation of cells for 30
26 The lithium acetate method gave significantly reproducible and minutes with the dye, following which, fluorescence intensity is
27 linear results (P<0.0001), as compared with direct measurement measured either directly (for intact cells), or using a cellular lysate.
28 (P=0.0005), sonication (P=0.1466) and bead beating (P=0.0028). It is known that shear stress and sonication can increase ROS
4–7
29 Key words: H2DCF-DA, Reactive Oxygen Species, Yeast production in vitro and this could interfere with the results. To
30 circumvent this problem, we developed a lithium acetate-SDS based
1. Introduction method to reduce the influence of externally generated ROS that
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could interfere with the results. In this paper, the various methods
32 The H2DCF-DA assay is a technique that is widely used to profile the of ROS measurement by DCF fluorescence have been compared to
33 oxidative status of living cells. It was initially developed by Brandt the Lithium Acetate-SDS method. The Lithium Acetate-SDS method
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34 et al in 1965 . The assay is based on cellular esterase action on seems efficient, less time consuming and gives consistent results for
35 H2DCF-DA to cleave acetate groups on the moiety, releasing an a large sample size.
36 intermediate H2DCF which reacts with ROS in its vicinity to form a
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37 fluorescent product, 2',7'-dichlorofluorescein (DCF) . H2DCF is 2. Materials and Methods
converted to the fluorescent DCF moiety by reaction with hydroxyl
38 •
radicals ( OH), peroxidase compounds I and II arising from catalysis The different methods used in this work were assessed in
39 of
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H2O2, NO2
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generated from the myeloperoxidase Saccharomyces cerevisiae MTCC 170. Yeast grown overnight was
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/H2O2/ NO2 system, hypochlorous acid (HOCl), and reactive species inoculated into fresh YEPD medium at room temperature, until the
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produced from peroxynitrite (ONOO /ONOOH) decomposition. OH
•
cells reached the exponential phase. Unless otherwise mentioned,
·− 7
42 or carbonate anion radicals ( CO3 ) are also known to be formed ~1.5×10 CFU/ml (OD660 = 0.9) cells were taken per sample for each
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43 during Peroxynitrite decomposition . Based on this, several experiment. H2DCF-DA was procured from Sigma Aldrich, India.
44 methods have been developed for quantitative and qualitative Fluorescence assays were performed using a Jasco
45 assessment of ROS production. Since ROS are short-lived, transient spectrofluorimeter FP8600 (Tokyo, Japan) interfaced with a
species and are difficult to estimate directly in vivo, only a few computer, using a 1cm path length quartz cuvette. Cells were taken
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methods that measure ROS have been widely accepted . The in ascending order and the linearity of H2DCF-DA fluorescence was
47 H2DCF-DA method gives an indirect measurement of the ROS assessed using one-way ANOVA followed by post-hoc linear trend
48 content in the cell, and therefore, any interference from procedures analysis between columns (P values < 0.05 were considered
49 employed in sample processing would yield high error values and significant). The cell number was kept constant to assess the
50 efficiency of the different protocols used. Yeast cultures were
51 harvested and centrifuged at 6000g for 10 minutes and the pellet
52 was washed twice in phosphate buffer (0.1M, pH 7.4). Cells were
53 incubated for 30 minutes with H2DCF-DA (10µM) in the same buffer
in the dark and then centrifuged. The washed pellet was taken for
54 further analysis for all the methods. The excitation and emission
55 wavelengths used for H2DCF-DA were 504nm and 524nm,
56 respectively.
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39 Figure 1. (A) Variation in ROS levels generated by different treatments. The number of yeast cells was 1.5 ×107 per treatment (B) Treatment of 0.5 ×107
40 yeast cells with Lithium acetate-SDS (C) Linearity of the methods employed: i. Direct fluorescence of a yeast cell suspension ii. Fluorescence of the extract
41 after Lithium acetate procedure iii. Fluorescence of lysate after sonication iv. Fluorescence of lysate after glass bead beating. All samples were diluted
sequentially from 1.5 to 0.5 ×107 cells the from stock culture
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43 3.4 Consistency of the Lithium Acetate method 4. Conclusions
44 12
The extraction method of Brennan and Schiestl gave an erratic
45 ROS signal due to the prolonged incubation time with SDS. ROS estimation using the H2DCF-DA method is error-prone due to
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46 However, treating the samples with lithium acetate and SDS for a several possible interferences . Yet, it is one of the most widely
47 little over 2 minutes did not generate much ROS (Figure 1B). Also, used methods. We suggest that sonication and enzymatic lysis be
48 the lithium acetate method gave consistent results (S.D=24.19) avoided in experiments that estimate ROS because the methods by
4–6
(Table 1). The extraction of the dye from the cells into the themselves are known to generate ROS . Bead beating and direct
49 fluorescence are indeed efficient methods in ROS estimation but
supernatant by agitation was uniform across all samples (Figure
50 1A), and thus, a large number of samples could be analysed within a are to be used with caution in experiments with a large sample size.
51 limited time frame. This method circumvents the undue increase in The Lithium Acetate-SDS method is seen to be both relatively
52 ROS generation during prolonged incubation periods. Also, this efficient and consistent and could be employed in estimating ROS
53 method is sensitive for a small number of cells and give readings for a large sample size. The Lithium acetate-SDS method does not
54 that can be highly correlated (P<0.0001, Figure 1C(ii)). Therefore, contribute to an erratic increase in ROS, because this method can
55 for a large sample size, this method could be employed as an be simultaneously performed for all the samples in a given
alternative to probe the oxidative status of yeast cells by avoiding experiment. This method gives relatively error free results, without
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superfluous ROS generation in the samples.
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11 References
12
13 1 R. Brandt and A. S. Keston, Anal. Biochem., 1965, 11, 6–9.
14 2 X. Chen, Z. Zhong, Z. Xu, L. Chen and Y. Wang, Free Radic.
15 Res., 2010, 44, 587–604.
16 3 B. Kalyanaraman, V. Darley-Usmar, K. J. A. Davies, P. A.
Dennery, H. J. Forman, M. B. Grisham, G. E. Mann, K.
17
Moore, L. J. Roberts and H. Ischiropoulos, Free Radic. Biol.
18 Med., 2012, 52, 1–6.
19 4 P. Riesz and T. Kondo, Free Radic. Biol. Med., 1992, 13,
20 247–270.
21 5 P. Riesz, D. Berdahl and C. L. Christman, Environ. Health
22 Perspect., 1985, 64, 233–252.
23 6 K. Milowska and T. Gabryelak, Biomol. Eng., 2007, 24, 263–
24 267.
7 S. Sahoo, K. K. Rao and G. K. Suraishkumar, Biotechnol.
25
Bioeng., 2006, 94, 118–127.
26 8 Y. Okai, K. Higashi-Okai, K. Machida, H. Nakamura, K.
27 Nakayama, K. Fujita, T. Tanaka, S. Otani and M. Taniguchi,
28 FEMS Microbiol. Lett., 2000, 185, 65–70.
29 9 E. Cabiscol, E. Piulats, P. Echave, E. Herrero and J. Ros, J.
30 Biol. Chem., 2000, 275, 27393–27398.
31 10 V. P. Martins, T. M. Dinamarco, F. M. Soriani, V. G. Tudella,
S. C. Oliveira, G. H. Goldman, C. Curti and S. A. Uyemura,
32
Eukaryot. Cell, 2011, 10, 237–48.
33 11 A. Horvath and H. Riezman, Yeast, 1994, 10, 1305–1310.
34 12 R. J. Brennan and R. H. Schiestl, Mutagenesis, 1997, 12,
35 215–220.
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32 A rapid method to extract DCF from yeast to measure its oxidative status by a fluorescent
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