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Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA)
使用氧化物探针2',7'-二氯二氟荧光素二乙酸酯(DCFH-DA)检测蓝细菌中活性氧物质(ROS)   

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Abstract

Reactive oxygen species (ROS) are cell signaling molecules synthesized inside the cells as a response to routine metabolic processes. In stress conditions such as ultraviolet radiation (UVR), ROS concentration increases several folds in the cells that become toxic for the cell survival. Here we present the method for in vivo detection of ROS by using an oxidant-sensing probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in cyanobacteria. This method provides reliable, simple, rapid and cost effective means for detection of ROS in cyanobacteria.

Keywords: Reactive oxygen species(活性氧), 2’,7’-Dichlorodihydrofluorescein diacetate(2',7'-二氯二氢荧光素二乙酸酯), Cyanobacteria(蓝藻), Ultraviolet radiation(紫外线辐射), Oxidative damage(氧化损伤)

Background

Cyanobacteria are the most ancient oxygenic photoautotrophs; they play an important role in the biomass production in both aquatic and terrestrial ecosystems and serve as source of various value-added products (Vaishampayan et al., 2001; Häder et al., 2007; Fischer, 2008). In recent years the depletion of the ozone layer has resulted in an increase in solar ultraviolet radiation (UVR) influx, which is harmful to all organisms residing on Earth including cyanobacteria (Holzinger and Lutz, 2006). The UVR harms cyanobacteria directly by acting on DNA/proteins or indirectly through oxidative damage from reactive oxygen species (ROS) (He and Häder, 2002). In plants, algal and mammalian cells various fluorescence and chemiluminescence methods have been used for detecting ROS (Crow, 1997; He and Häder, 2002; Soh, 2006; Wu et al., 2007; Palomero et al., 2008).

2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) is a non-fluorescent, cell-permeable dye which is hydrolyzed intracellularly into its polar, but non-fluorescent form DCFH on the action of cellular esterases and thus is retained in the cell. Oxidation of DCFH by the action of intracellular ROS and other peroxides turns the molecule into its highly fluorescent form 2’,7’-dichlorofluorescein (DCF) that can be detected by various fluorescent methods (He and Häder, 2002; Rastogi et al., 2010; Singh et al., 2014) (Figure 1). Although DCFH-DA is widely used for the detection of ROS, it should be noted, however, that the dye cannot be used as an indicator for a specific form of ROS (Marchesi et al., 1999).


Figure 1. Mechanism of action of DCFH-DA probe inside the cell (Adapted from He and Häder, 2002)

Materials and Reagents

  1. 2 ml RNase, DNase free microcentrifuge tube (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM12425 )
  2. Glass microscope slides (Fisher Scientific, catalog number: 12-544-4 )
  3. Glass microscope coverslips (Fisher Scientific, catalog number: S17525B )
  4. Millipore membrane filter (EMD Millipore, catalog number: HAWP04700 )
  5. Cuvette (fluorescence spectroscopy) 3 ml (Hellma, catalog number: 101-QS )
  6. Cyanobacterial cells e.g., Nostoc sp. strain HKAR-2
    Note: Nostoc sp. strain HKAR-2, an autotrophic, filamentous and heterocystous cyanobacterium, was grown under axenic conditions in nitrogen-free liquid BGA medium (Safferman and Morris, 1964) at 20 ± 2 °C under continuous white light (12 ± 2 Wm-2) to an OD750 of 0.8 to 0.9 (exponential growth phase) which was measured using quartz cuvette in a spectrophotometer.
  7. Nail varnish (Lakme)
  8. Potassium phosphate dibasic anhydrous (K2HPO4)
  9. Potassium phosphate monobasic (KH2PO4)
  10. 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, catalog number: D6883 )
  11. 100% ethanol (Sigma-Aldrich, catalog number: 459836 )
  12. 50 mM phosphate buffer (see Recipes)
  13. 2 mM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) stock solution (see Recipes)

Equipment

  1. Glass Petri-dishes (Corning, catalog number: 3160-102 )
  2. Measuring cylinder
  3. 295 nm UV cut-off filter (Ultraphan, Digefra, Munich, Germany) to facilitate the desired wavebands of UV-B (280-315 nm), UV-A (315-400 nm) and PAR (400-700 nm)
  4. UV-treatment chamber fitted with UV-B (Philips Ultraviolet-B TL 40 W: 12, Philips Lighting, model: TL 40W/12 RS SLV/25 ), UV-A (Philips Ultraviolet-A TL 40 W: 12, Philips Lighting, model: TL-K 40W/10-R UV-A ) and PAR (55.08  ±  9.18 μmol m-2 sec-1) (OSRAM L 36 W: 32 Lumilux de luxe warm white and Radium NL 36 W: 26 Universal white, Germany) lamps
  5. Glass rod (Fisher Scientific, catalog number: 11-380A )
  6. Magnetic stirrer (REMI ELECTROTECHNIK, model: 2 MLH )
  7. Refrigerated centrifuge (REMI ELECTROTECHNIK, model: CM-12 PLUS )
  8. Shaker
  9. Fluorescence microscope (Nikon eclipse Ni fluorescence microscope processed by NIS Elements (BR))
  10. Fluorescence spectrophotometer (Agilent Technologies, model: Cary Eclipse )
  11. Spectrophotometer (Hitachi High-Technologies, model: U-2900 , Double beam spectrophotometer)
  12. Quartz cuvette (3.5 ml) (Cole-Parmer, JENWAY, catalog number: 035 028 )

Software

  1. NIS-Elements (BR) imaging software (Nikon)
  2. SigmaPlot 11 software

Procedure

A flow chart of the sample preparation is shown in Figure 2.


Figure 2. Flow chart showing various steps involved in the protocol

  1. Transfer 100 ml of cyanobacterial culture to sterile glass Petri-dishes with the help of a measuring cylinder. Cover the Petri dishes with a 295 nm UV cut-off filter and transfer them to a UV light treatment chamber.
  2. Irradiate the cyanobacteria with UV-A, UV-B and PAR and maintain the temperature of the chamber at 25 ± 2 °C to avoid a heating effect. Mix the culture with a glass rod at regular intervals or use magnetic stirrer (15-20 rpm) to avoid self-shading of the cells.
  3. After desired time intervals (hereafter 12 h and 24 h), take 5 ml of sample and harvest cells by centrifugation at 9,050 x g for 20 min at room temperature.
  4. Resuspend the cell pellet in 1 ml phosphate buffer (see Recipes).
  5. Add 2.5 µl of 2 mM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) solubilized in ethanol (see Recipes) to the sample mixture.
  6. Incubate the sample mixture on a shaker (15 rpm) at room temperature in the dark for 1 h.
  7. After 1 h incubation
    1. For fluorescence microscopy: Take a clean glass slide and add 30 µl of culture. Cover the cells with a glass cover slip. Seal the slide with nail varnish to avoid drying out. Cells were visualized under a fluorescence microscope using an excitation wavelength of 488 nm and emission was detected in the range of 500-600 nm (Figure 3).


      Figure 3. Fluorescence images of UV-A + UV-B + PAR exposed Nostoc sp. strain HKAR-2 showing green DCF fluorescence after reaction with ROS. Negative control (containing DCFH-DA only; showing the basal level of fluorescence) (A); fluorescence at 0 h (B); 12 h UV-A + UV-B + PAR exposure (C); 24 h UV-A + UV-B + PAR exposure (D). PAR: Photosynthetically active radiation. Scale bar =10 µm.

    2. For fluorescence spectroscopy: 3 ml of liquid sample was added to a cuvette for fluorescence spectrophotometric analysis (Figure 4). The cuvette was placed in fluorescence spectrophotometer and the sample was excited at 485 nm. Emission was recorded in the range of 500-600 nm. The exposure time was limited to 600 msec to reduce the damage of cells. Fluorescence was measured in terms of emitted fluorescence intensity after different durations of stress exposure.


      Figure 4. Fluorescence intensity of DCF after reaction with ROS generated due to varying duration of UV-A + UV-B + PAR exposure in Nostoc sp. strain HKAR-2 (means ± SD, n = 3). Control: Untreated sample. PAR: Photosynthetically active radiation.

Data analysis

The UVR irradiated cells were analyzed using a Nikon Eclipse Ni fluorescence microscope processed by NIS-Elements (BR) imaging software. The microscope was equipped with the following filter set: UV: (DAPI) EX 340 nm EM 488 nm, blue: (FITC) EX 495 nm EM 510 nm and green: (PI 550) EX 550 nm EM 650 nm. Cells were imaged in the epifluorescence mode with a 20x objective lens. The image analysis was performed by NIS-Elements (BR) imaging software provided by Nikon and images were saved in JPEG format. In addition, the fluorescence of the samples was measured by a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies) with an excitation wavelength of 485 nm and an emission band between 500 and 600 nm. The data of the fluorescence spectra were exported to excel and the fluorescence intensity values at 525 nm were extracted. A bar diagram was plotted with SigmaPlot 11 software. All fluorescence measurements were performed at room temperature. All results are presented as mean values of three replicates for fluorescence spectrophotometer analysis and random sites of filaments were used for fluorescence microscopy. All data were analysed by one-way analysis of variance (Brown, 2005). Once a significant difference was detected post hoc multiple comparisons were made by using the Tukey test. The level of significance was set at 0.05 for all tests. All statistical analyses were performed by using SigmaPlot 11 software.

Notes

  1. Direct exposure of light to UV treated samples should be avoided.
  2. The stock solution of 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) once prepared, was kept in -20 °C for further use and direct exposure to light should be avoided (it remains stable for more than 3 months).
  3. All solutions were filtered through 0.25 µm size Millipore membrane filter before use.

Recipes

  1. 50 mM phosphate buffer (400 ml)
    8.7 g K2HPO4
    6.8 g KH2PO4
    Adjust the pH to 7.00
    Filter through 0.25 µm size Millipore membrane filter before use
  2. 2 mM (w/v) 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) stock solution
    Dissolve 0.974 mg of 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in 1 ml of absolute ethanol

Acknowledgments

Rajneesh and Jainendra Pathak are thankful to the Department of Biotechnology (DBT-JRF/13/AL/143/2158) and the Council of Scientific and Industrial Research (09/013/0515/2013-EMR-I), New Delhi, India, respectively, for the financial support in the form of fellowships. SP Singh acknowledges the DST-SERB and UGC for Early Career Research Award and UGC Start-Up Research Grant, respectively. We are also thankful to the Interdisciplinary School of Life Sciences (ISLS), BHU, Varanasi, India, for providing access to the fluorescence microscopy facility. This protocol was adapted from procedures published by Rastogi et al., 2010.

References

  1. Brown, A. M. (2005). A new software for carrying out one-way ANOVA post hoc tests. Comput Methods Programs Biomed 79(1): 89-95.
  2. Crow, J. P. (1997). Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide 1(2): 145-157.
  3. Fischer, W. F. (2008). Life before the rise of oxygen. Nature 455: 1051-1052.
  4. Häder, D. P., Kumar, H. D., Smith, R. C. and Worrest, R. C. (2007). Effects of solar UV radiation on aquatic ecosystems and interactions with climate change. Photochem Photobiol Sci 6(3): 267-285.
  5. He, Y. Y. and Häder, D. P. (2002). UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine. J Photochem Photobiol B 66(2): 115-124.
  6. Holzinger, A. and Lutz, C. (2006). Algae and UV irradiation: effects on ultrastructure and related metabolic functions. Micron 37(3): 190-207.
  7. Marchesi, E., Rota, C., Fann, Y. C., Chignell, C. F. and Mason, R. P. (1999). Photoreduction of the fluorescent dye 2’-7’-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements. Free RadicBiol Med 26(1-2): 148-161.
  8. Palomero, J., Pye, D., Kabayo, T., Spiller, D. G. and Jackson, M. J. (2008). In situ detection and measurement of intracellular reactive oxygen species in single isolated mature skeletal muscle fibers by real time fluorescence microscopy. Antioxid Redox Signal 10(8): 1463-1474.
  9. Rastogi, R. P., Singh, S. P., Häder, D. P. and Sinha, R. P. (2010). Detection of reactive oxygen species (ROS) by the oxidant-sensing probe 2’,7’-dichlorodihydrofluorescein diacetate in the cyanobacterium Anabaena variabilis PCC 7937. Biochem Biophys Res Commun 397(3): 603-607.
  10. Safferman, R. S. and Morris, M E. (1964). Growth characteristics of the blue-green algal virus LPP1. J Bacteriol 88: 771-775.
  11. Singh, S. P., Rastogi, R. P., Hader, D. P. and Sinha, R. P. (2014). Temporal dynamics of ROS biogenesis under simulated solar radiation in the cyanobacterium Anabaena variabilis PCC 7937. Protoplasma 251(5): 1223-1230.
  12. Soh, N. (2006). Recent advances in fluorescent probes for the detection of reactive oxygen species. Anal Bioanal Chem 386(3): 532-543.
  13. Vaishampayan, A., Sinha, R. P., Häder, D. P., Dey, T., Gupta, A. K., Bhan, U. and Rao, A. L. (2001). Cyanobacterial biofertilizers in rice agriculture. Bot Rev 67: 453-516.
  14. Wu, T. T. Chen, K. and Keaney, J. F. (2007). Use of 2’,7’-dichlorodihydrofluorescein diacetate in an easy and quantifiable assay for hypochlorous acid oxidation. FASEB J 21: 882-889.

简介

活性氧(ROS)是细胞内合成的细胞信号分子,作为对常规代谢过程的反应。 在紫外线照射(UVR)等应激条件下,ROS浓度在细胞中增加数倍,对细胞存活有毒性。 在这里,我们介绍了通过使用蓝细菌中的氧化剂感测探针2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)来体内检测ROS的方法。 该方法提供可靠,简单,快速和成本有效的检测蓝细菌中的ROS的方法。
【背景】蓝藻是最古老的含氧光合自养体;它们在水生和陆地生态系统的生物量生产中发挥重要作用,并作为各种增值产品的来源(Vaishampayan等,2001;Häderet al。,2007; Fischer,2008)。近年来,臭氧层的消耗导致太阳紫外线辐射(UVR)涌入增加,这对所有存在于地球上的生物(包括蓝细菌)都是有害的(Holzinger和Lutz,2006)。 UVR通过作用于DNA /蛋白质或间接通过活性氧(ROS)的氧化损伤直接伤害蓝细菌(He和Häder,2002)。在植物,藻类和哺乳动物细胞中,已经使用各种荧光和化学发光方法检测ROS(Crow,1997; He和Häder,2002; Soh,2006; Wu et al。,2007; Palomero et al。,2008)。
  2',7'-二氯二氟荧光素二乙酸酯(DCFH-DA)是一种非荧光,细胞可渗透的染料,其在细胞酯酶的作用下在细胞内水解成其极性但非荧光形式的DCFH,因此保留在细胞中。通过细胞内ROS和其他过氧化物的作用氧化DCFH使分子变成其可通过各种荧光方法检测的2',7'-二氯荧光素(DCF)的高度荧光形式(He和Häder,2002; Rastogi等人, 2010; Singh et al。,2014)(图1)。尽管DCFH-DA广泛用于检测ROS,但应注意的是染料不能用作特定形式的ROS的指示剂(Marchesi等,1999)。

关键字:活性氧, 2',7'-二氯二氢荧光素二乙酸酯, 蓝藻, 紫外线辐射, 氧化损伤

材料和试剂

  1. 2ml RNase,无DNA酶的微量离心管(Thermo Fisher Scientific,Invitrogen TM,目录号:AM12425)
  2. 玻璃显微镜载玻片(Fisher Scientific,目录号:12-544-4)
  3. 玻璃显微镜盖玻片(Fisher Scientific,目录号:S17525B)
  4. 微孔膜过滤器(EMD Millipore,目录号:HAWP04700)
  5. 比浊蛋白(荧光光谱)3ml(Hellma,目录号:101-QS)
  6. 蓝藻细胞,例如,,Nostoc sp。应变HKAR-2
    注意:Nostoc sp。在无氮液体BGA培养基(Safferman和Morris,1964)的无菌条件下,在连续白光(12±2Wm 20±2℃)下,在无菌条件下生长HKAR-2菌株HKAR-2 -2 )到使用分光光度计中使用石英比色杯测量的0.8至0.9(指数生长期)的OD 750。
  7. 指甲油(Lakme)
  8. 无水磷酸氢二钾(K 2 HPO 4)
  9. 磷酸二氢钾(KH 2 PO 4)
  10. 2',7'-二氯二氟荧光素二乙酸酯(DCFH-DA)(Sigma-Aldrich,目录号:D6883)
  11. 100%乙醇(Sigma-Aldrich,目录号:459836)
  12. 50 mM磷酸盐缓冲液(见配方)
  13. 2mM 2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)储备溶液(参见食谱)

设备

  1. 玻璃培养皿(康宁,目录号:3160-102)
  2. 测量缸
  3. 295nm UV截止滤光片(Ultraphan,Digefra,Munich,Germany),以促进UV-B(280-315nm),UV-A(315-400nm)和PAR(400-700nm)的期望波段br />
  4. UV-A(Philips Ultraviolet-A TL 40 W:12,Philips Ultraviolet-B TL 40 W:12,Philips Lighting,型号:TL 40W / 12 RS SLV / 25)照明,型号:TL-K 40W / 10-R UV-A)和PAR(55.08±9.18μmol,m -2 sec -1)(OSRAM L 36 W: 32 Lumilux de luxe暖白和镭NL 36 W:26通用白,德国)灯
  5. 玻璃棒(Fisher Scientific,目录号:11-380A)
  6. 磁力搅拌器(REMI ELECTROTECHNIK,型号:2 MLH)
  7. 冷冻离心机(REMI ELECTROTECHNIK,型号:CM-12 PLUS)
  8. 振动器
  9. 荧光显微镜(NIS Elements(BR)处理的尼康日光镍荧光显微镜)
  10. 荧光分光光度计(Agilent Technologies,型号:Cary Eclipse)
  11. 分光光度计(日立高科技,型号:U-2900,双光束分光光度计)
  12. 石英比色皿(3.5ml)(Cole-Parmer,JENWAY,目录号:035 028)

软件

  1. NIS-Elements(BR)成像软件(尼康)
  2. SigmaPlot 11软件

程序

样品制备的流程图如图2所示

图2.显示协议中涉及的各种步骤的流程图

  1. 借助于量筒,将100ml蓝藻培养物转移到无菌玻璃培养皿中。用295 nm紫外线截止滤光片覆盖培养皿,并将其转移到紫外光处理室。
  2. 用UV-A,UV-B和PAR辐射蓝细菌,并将室温保持在25±2℃,以避免加热效应。将培养物与玻璃棒定期混合,或使用磁力搅拌器(15-20rpm)以避免细胞的自阴影。
  3. 在所需的时间间隔(在12小时和24小时后),通过在室温下以9,050×g离心20分钟,取5ml样品并收获细胞。
  4. 将细胞沉淀重悬于1ml磷酸盐缓冲液中(参见食谱)
  5. 加入2.5μl溶于乙醇的2mM 2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)(参见食谱)至样品混合物。
  6. 在室温下在黑暗中将样品混合物在振荡器(15rpm)上孵育1小时。
  7. 孵育1小时后
    1. 对于荧光显微镜:取一张干净的载玻片并加入30微升的培养物。用玻璃盖板盖住电池。用指甲油密封滑块,以免干燥。在荧光显微镜下使用488nm的激发波长显现细胞,并且在500-600nm的范围内检测到发射(图3)。


      图3. UV-A + UV-B + PAR暴露的Nostoc 的荧光图像。应答HKAR-2与ROS反应后显示绿色DCF荧光。阴性对照(仅含DCFH-DA;显示基础荧光水平)(A); 0小时荧光(B); 12小时UV-A + UV-B + PAR曝光(C); 24小时UV-A + UV-B + PAR曝光(D)。 PAR:光合有效辐射。比例尺= 10微米。

    2. 对于荧光光谱:将3ml液体样品加入到比色皿中进行荧光分光光度分析(图4)。将试管置于荧光分光光度计中,样品在485nm激发。记录在500-600nm的范围内。曝光时间限制在600毫秒,以减少细胞的损伤。在不同持续时间的应激暴露后,根据发射的荧光强度测量荧光

      图4.由于在Nostoc sp中UV-A + UV-B + PAR暴露的持续时间变化而产生的ROS反应后的DCF的荧光强度。菌株HKAR-2 (表示±SD,n = 3)。对照:未处理样品。 PAR:光合有效辐射。

数据分析

使用由NIS-Elements(BR)成像软件处理的Nikon eclipse Ni荧光显微镜分析UVR照射的细胞。显微镜配备以下滤光片:UV:(DAPI)EX 340nm EM 488nm,蓝色:(FITC)EX 495nm EM 510nm和绿色:(PI 550)EX 550nm EM 650nm。用20倍物镜在细胞荧光模式下成像细胞。图像分析由尼康提供的NIS-Elements(BR)成像软件进行,图像以JPEG格式保存。此外,通过荧光分光光度计(Cary eclipse,Agilent Technologies)测量样品的荧光,激发波长为485nm,发射带为500至600nm。将荧光光谱数据输出至excel,并提取525nm处的荧光强度值。使用SigmaPlot 11软件绘制条形图。所有荧光测量均在室温下进行。所有结果以荧光分光光度计分析的三次重复的平均值表示,长丝的随机位点用于荧光显微镜。所有数据通过单因素方差分析(Brown,2005)。一旦检测到显着性差异,则使用Tukey检验进行事后多重比较。所有测试的显着性水平为0.05。所有统计分析均采用SigmaPlot 11软件进行。

笔记

  1. 应避免将光直接暴露于紫外线处理的样品。
  2. 将一次制备的2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)的储备溶液保持在-20℃进一步使用,应避免直接暴露于光照(其保持稳定3个月以上)。 br />
  3. 所有溶液在使用前通过0.25μm尺寸的Millipore膜过滤器过滤

食谱

  1. 50毫克磷酸盐缓冲液(400毫升)
    8.7g K 2 HPO 4
    6.8g KH 2 PO 4
    将pH调节至7.00
    使用前过滤0.25μm尺寸的Millipore膜过滤器
  2. 2mM(w / v)2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)储备溶液
    将0.974mg 2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)溶于1ml无水乙醇中,

致谢

Rajneesh和Jainendra Pathak感谢生物科技部(DBT-JRF / 13 / AL / 143/2158)和科学与工业研究理事会(09/013/0515/2013-EMR-I),印度新德里分别以奖学金的形式提供财政支持。 SP Singh分别承认了DST-SERB和UGC的早期职业研究奖和UGC创业研究奖学金。我们也感谢生物科学学院(ISLS),BHU,印度瓦拉纳西,提供荧光显微镜设备的访问。该方案根据Rastogi等人,2010年出版的方法改编。

参考

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引用:Rajneesh, , Pathak, J., Chatterjee, A., Singh, S. P. and Sinha, R. P. (2017). Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA). Bio-protocol 7(17): e2545. DOI: 10.21769/BioProtoc.2545.
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