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Measurement of Intracellular ROS in Caenorhabditis elegans Using 2’,7’-Dichlorodihydrofluorescein Diacetate
使用2',7'-二氯二氢荧光素二乙酸盐测定秀丽隐杆线虫细胞内ROS   

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Scientific Reports
Oct 2017

Abstract

Reactive oxygen species (ROS) are generated during normal metabolic processes under aerobic conditions. Since ROS production initiates harmful radical chain reactions on cellular macromolecules, including lipid peroxidation, DNA mutation, and protein denaturation, it has been implicated in a wide spectrum of diseases such as cancer, cardiovascular disease, ischemia-reperfusion and aging. Over the past several decades, antioxidants have received explosive attention regarding their protective potential against these deleterious reactions. Accordingly, many analytical methodologies have been developed for the evaluation of the antioxidant capacity of compounds or complex biological samples. Herein, we introduce a simple and convenient method to detect in vivo intracellular ROS levels photometrically in Caenorhabditis elegans using 2’,7’-dichlorofluorescein diacetate (H2DCFDA), a cell permeant tracer.

Keywords: Reactive oxygen species (活性氧族), 2’,7’-Dichlorofluorescein diacetate (2',7'-二氯荧光素二乙酸盐), C. elegans (秀丽隐杆线虫), Antioxidant (抗氧化剂)

Background

In situ detection of intracellular reactive oxygen species (ROS) levels in the living organism using fluorescent probe 2’,7’-dichlorofluorescein diacetate (H2DCFDA) has been broadly performed by those researchers who work in the field of oxidative stress and related diseases. The non-polar and non-ionic probe, H2DCFDA, can easily penetrate the cellular membrane and is enzymatically deacetylated by esterases. This biochemical reaction turns H2DCFDA into the non-fluorescent compound H2DCF which is then rapidly oxidized to highly fluorescent 2’,7’-dichlorofluorescein (DCF) in the presence of ROS (Figure 1A). Therefore, fluorescence signals from H2DCFDA probe demonstrate important information for the quantification of ROS at single cell level (Labuschagne and Brenkman, 2013). The Caenorhabditis elegans model system provides an excellent in vivo experimental environment for evaluating molecular mechanisms of ROS pathophysiology due to their short lifespan, simplicity, and ease of genetic manipulation (Labuschagne and Brenkman, 2013; Miranda-Vizuete and Veal, 2017; Yoon et al., 2017) (Figure 1B). Here, we describe a simple protocol to measure the levels of time-course ROS generation in C. elegans using H2DCFDA under normal and heat- or chemically-induced oxidative stress conditions. Using this protocol, we determined the effects of H2DCFDA concentration and number of tested worms on DCF fluorescence signal (Figure 2).


Figure 1. ROS detection. A. Production of florescent DCF by intracellular ROS. B. Measurement of intracellular ROS using molecular probe (H2DCFDA) in C. elegans.


Figure 2. Change in DCF fluorescence signals depends on experimental conditions. A. The expression of gcs-1(promoter)::GFP (oxidative stress marker) transgene by oxidative stress. Heat (30 °C for 2 h) induced the expression of gcs-1(promoter)::GFP transgene in the intestines of live nematodes. B. Concentration-response curves were plotted in terms of the mean value of DCF fluorescence signals induced by 12.5, 25, 50, and 100 μM of H2DCFDA using 50 nematodes for each experiment. C. Changes in DCF fluorescence signals depending on the number of tested nematodes (10, 20, 50, and 100) were assessed using 25 μM of H2DCFDA.

Materials and Reagents

  1. 1.5 ml microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
  2. Conical tube, 15 ml (Corning, Falcon®, catalog number: 352096 )
  3. Slide glass (VWR, catalog number: 48300-025 )
  4. 96-well microplate, black (SPL Life Sciences, catalog number: 30496 )
  5. 60 mm Petri dish (SPL Life Sciences, catalog number: 10060 )
  6. Platinum wire, 0.2 mm (Alfa Aesar, catalog number: 45093 )
  7. Caenorhabditis elegans strain, wild-type [N2] (Caenorhabditis Genetics Center, University of Minnesota: https://cgc.umn.edu)
  8. Escherichia coli OP50 strain (Caenorhabditis Genetic Center)
  9. Distilled water
  10. 2’,7’-Dichlorofluorescein diacetate (Sigma-Aldrich, catalog number: D6883 )
  11. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  12. Methyl viologen dichloride hydrate (Merck, catalog number: 856177 )
  13. Cholesterol (Sigma-Aldrich, catalog number: C8667 )
  14. Ethanol (Sigma-Aldrich, catalog number: E7023 )
  15. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  16. Bacto-peptone (BD, BactoTM, catalog number: 211677 )
  17. Agar (Sigma-Aldrich, catalog number: A1296 )
  18. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  19. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  20. Potassium phosphate, dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  21. Potassium phosphate, monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 795488 )
  22. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  23. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  24. Sodium hypochlorite (NaClO) (Sigma-Aldrich, catalog number: 425044 )
  25. 5 mg/ml cholesterol (see Recipes)
  26. Nematode growth medium (NGM) agar plates (see Recipes)
  27. M9 buffer (see Recipes)
  28. Bleaching solution (see Recipes)

Equipment

  1. Shaking incubator (Benchtop Shaking Incubator, VWR, model: Model 1570 )
  2. Micropipette (VWR International)
  3. Incubators for stable temperature (VWR SIGNATURE 20-cuft Model 2020 B.O.D. -10 °C to 45 °C Low Temp Incubator) (VWR, Advanced Instruments, model: Model 2020 )
  4. Tabletop centrifuge (VWR, model: VWR® Mini Centrifuge C1413V )
  5. Dissecting stereomicroscope (Stereo Microscope with Apochromatic Optics Leica S8 APO) (Leica Microsystems, model: Leica S8 APO )
  6. Fluorophotometer (Promega, model: GloMax®-Multi Microplate Multimode Reader )
  7. Vortex mixer (Standard Heavy-Duty Vortex Mixer, VWR, catalog number: 97043-562 )
  8. Autoclave (Hanshin Medical, model: HS-2321SD )

Software

  1. Microsoft Office 2017 Excel (Microsoft Corporation, Redmond, USA)
  2. IBM SPSS statistics 24 (IBM Corporation, New York, USA)

Procedure

  1. Measuring intracellular ROS of nematodes under normal culture conditions
    1. Preparation of a synchronized nematode population
      1. Wash a plate containing full adults and eggs with 1 ml of M9 buffer.
      2. Transfer nematode suspension into a microcentrifuge tube.
      3. Spin down at 3,884 x g (rcf; 8,500 rpm) for 1 min and remove the supernatant.
      4. Add 200 μl of Distilled Water (D.W.) and 800 μl of bleaching solution.
      5. Wait until over 90% of nematodes have lysed and several embryos are released [please see Section 3.2 in Bianchi and Driscoll (2006)].
        Note: Check for lysis every 30 sec after 3 min. Vortexing at maximum speed for about 2-3 sec helps with bleach lysis.
      6. Spin down at 3,884 x g (rcf; 8,500 rpm) for 1 min and remove the supernatant.
        Note: Be careful not to disturb the pellet.
      7. Wash three times with M9 buffer (1 ml each): Spin down at 137 x g (rcf; 1,000 rpm) for 1 min and remove the supernatant.
      8. Transfer embryos with 1 ml of M9 buffer to a conical tube containing 1 ml of M9 (total 2 ml).
      9. Incubate overnight at 20 °C with shaking at 30 rpm to allow L1 larvae to hatch.
    2. Counting animals and preparation of nematode solution
      1. Resuspend the larva pellet and spread 10 μl of the suspension onto a glass slide. Do this in triplicate.
      2. Count the number of nematodes using a dissecting stereomicroscope and calculate the average number of L1 larvae in each drop.
      3. Add an appropriate volume of M9 buffer to dilute the larva suspension.
      4. Shake gently and count again.
      5. Repeat Steps A2b-A2d until the number of nematodes arrives to around 50 per 10 μl.
    3. Preparation of H2DCFDA reaction solution
      1. Prepare a concentrated stock (50 mM) by dissolving 24.365 mg of H2DCFDA in 1 ml of cell culture grade DMSO.
      2. Split into 100 μl aliquots in black microcentrifuge tubes and store at -20 °C (stable for at least 3 months).
      3. Dilute stock solution to 50 μM with M9 buffer as required (volume required = number of test wells x 50 μl).
        Note: Working solution should be prepared shortly before use.
    4. Determination of intracellular ROS levels using a fluorophotometer
      1. Turn on the fluorophotometer and set plate settings.
      2. Add 40 μl of M9 buffer into the wells of a black 96-well plate.
      3. Add 10 μl of nematode solution (prepared in Step A2) into each well.
      4. Add 50 μl of H2DCFDA reaction solution to a final concentration of 25 μM.
      5. Prepare blank wells by adding 50 μl of M9 and 50 μl of H2DCFDA reaction solution.
      6. Immediately, insert the plate into the fluorophotometer and let gently shake for 30 sec.
      7. Quantify the fluorescence intensity at an excitation wavelength of 490 nm and an emission wavelength of 510-570 nm.
      8. Measure fluorescence signal every hour (maximum 6 h) and export raw data through USB port (see Figure 2A).
    Note: All steps should be done at RT.

  2. Measuring intracellular ROS of nematodes under oxidative stress conditions
    1. Paraquat-induced oxidative stress
      1. Make 50 mM paraquat solution by dissolving methyl viologen dichloride hydrate (paraquat) powder in D.W.
      2. Prepare arrested L1 larvae as described in Step A1.
      3. Add 1 part 50 mM paraquat to 9 parts L1-containing M9 buffer (1:10 dilution) and incubate at 20 °C for 2 h with shaking at 30 rpm in the shaking incubator.
      4. Wash three times with M9 buffer (1 ml each): Spin down at 137 x g (rcf; 1,000 rpm) for 1 min and remove the supernatant.
      5. Prepare nematode solution as described in Step A2.
      6. Quantify intracellular ROS levels as described in Steps A3 and A4.
    2. Heat-induced oxidative stress
      1. Prepare arrested L1 larvae and nematode solution as described in Steps A1 and A2.
      2. Incubate nematodes at 30 °C for 2 h with shaking at 30 rpm in the shaking incubator.
      3. Quantify intracellular ROS levels as described in Steps A3 and A4.
        Note: Maintain the fluorophotometer temperature at 30 °C throughout the measurement.

Data analysis

  1. For each condition, use at least 20 animals to obtain accurate results.
  2. Each experiment should be performed at least three independent times.
  3. Blank-subtracted raw data (DCF fluorescence level) are normalized to those in wild-type (or untreated) nematodes and are presented as mean ± standard deviation using Microsoft Office 2017 Excel software.
  4. The one-way ANOVA with Tukey’s post-hoc test is carried out using IBM SPSS statistics 24 to analyze the statistical significance of differences between groups.

Notes

  1. As is the case with many other fluorescent probes, H2DCFDA is photoactive, and thus, minimal exposure to light when handling is vital.
  2. Whole nematodes should be used instead of lysed nematodes when measuring intracellular ROS using H2DCFDA in C. elegans. Lysis causes disruption of outer cuticle and internal membranes followed by the release of intracellular metal ions such as iron. Since free iron can produce ROS by itself through the Fenton reaction, ROS levels may be overestimated in lysed nematodes compared to measurements in the whole nematode.
  3. Since H2DCFDA readily diffuses into cells until it reaches equilibrium, a time course of increased basal fluorescence signal can be detected. The gradual increasing tendency is shifted to steep upcurve at the time point of animal death. Therefore, taking measurements within 4 or 6 h after starting experiment is appropriate.
  4. Adult nematodes (age-synchronized) can also be used to test antioxidant capacity of compounds under oxidative stress conditions with minor modifications. Using a small number of nematodes is recommended (10-20 worms/well). Also, increasing paraquat concentration is required to induce pronounced oxidative stress in adult nematodes (over 20 mM, final concentration). However, a relatively low concentration of paraquat should be given to aged nematodes.

Recipes

  1. 5 mg/ml cholesterol
    5 mg cholesterol powder is dissolved in 1 ml of 100% ethanol and stored at 4 °C
  2. Nematode growth medium (NGM) agar plates (per L)
    3 g of NaCl
    2.5 g of peptone
    17 g of agar
    1 ml of cholesterol solution (5 mg/ml)
    1 ml of CaCl2 (1 M)
    1 ml of MgSO4 (1 M)
    25 ml of potassium phosphate (1 M, pH 6.0)
    975 ml of distilled water
    Autoclave and cool to 50 °C
    Pour 10 ml of NGM agar into 60 mm Petri dish and let harden for 1 h
  3. M9 buffer
    3 g of KH2PO4
    6 g of Na2HPO4
    5 g of NaCl
    1 ml of MgSO4 (1 M)
    Make up to 1 L with distilled water
    Autoclave
  4. Bleaching solution
    6.25 ml of 4 M NaOH
    2.5 ml of NaOCl
    50 ml of M9 buffer
    Prepare freshly before use

Acknowledgments

This work was supported in part by the National Research Foundation of South Korea (NRF-2017R1C1B5015695) to DSC, Brody Brothers Endowment Grant (21602-664261), NIH (1R15GM112174-01A1), NSF (MCB1714264) to M-H.L. The Caenorhabditis Genetics Center (CGC) is supported by the National Institutes of Health – Office of Research Infrastructure Programs (P40 OD010440). This protocol has been adapted from Yoon et al., 2017. The authors have declared that no competing interests exist.

References

  1. Bianchi, L. and Driscoll, M. (2006). Culture of embryonic C. elegans cells for electrophysiological and pharmacological analyses. WormBook 1-15.
  2. Labuschagne, C. F. and Brenkman, A. B. (2013). Current methods in quantifying ROS and oxidative damage in Caenorhabditis elegans and other model organism of aging. Ageing Res Rev 12(4): 918-930.
  3. Miranda-Vizuete, A. and Veal, E. A. (2017). Caenorhabditis elegans as a model for understanding ROS function in physiology and disease. Redox Biol 11: 708-714.
  4. Yoon, D. S., Choi, Y., Cha, D. S., Zhang, P., Choi, S. M., Alfhili, M. A., Polli, J. R., Pendergrass, D., Taki, F. A., Kapalavavi, B., Pan, X., Zhang, B., Blackwell, T. K., Lee, J. W. and Lee, M. H. (2017). Triclosan disrupts SKN-1/Nrf2-mediated oxidative stress response in C. elegans and human mesenchymal stem cells. Sci Rep 7(1): 12592.

简介

在有氧条件下的正常代谢过程中产生活性氧物质(ROS)。 由于ROS产生会引起细胞大分子的有害自由基连锁反应,包括脂质过氧化反应,DNA突变和蛋白质变性,因此它已涉及多种疾病,如癌症,心血管疾病,缺血再灌注和衰老。 在过去的几十年中,抗氧化剂已经受到爆炸性的关注,因为它们对这些有害反应具有保护作用。 因此,已经开发了许多分析方法用于评估化合物或复杂生物样品的抗氧化能力。 在此,我们介绍了使用2',7'-二氯荧光素二乙酸盐(H 2),在线虫体内用光度计检测体内细胞内ROS水平的简单方便的方法。 DCFDA),一种细胞渗透性示踪剂。

【背景】已经广泛地使用荧光探针2',7'-二氯荧光素二乙酸酯(H 2 DCFDA)原位检测活体内细胞内活性氧簇(ROS)水平由在氧化应激和相关疾病领域工作的研究人员提供。非极性和非离子型探针H 2 DCFDA可以容易地穿透细胞膜并且被酯酶酶促脱乙酰化。该生物化学反应将H 2 DCFDA转化成非荧光化合物H 2 DCF,然后将其迅速氧化成高度荧光的2',7'-二氯荧光素(DCF) ROS的存在(图1A)。因此,来自H 2 DCFDA探针的荧光信号证明了在单细胞水平上定量ROS的重要信息(Labuschagne和Brenkman,2013)。秀丽隐杆线虫模型系统提供了用于评估ROS病理生理学的分子机制的极好的体内实验环境,因为它们的寿命短,简单并且易于遗传操作(Labuschagne和Brenkman,2013; Miranda-Vizuete和Veal,2017; Yoon et al。,2017)(图1B)。在这里,我们描述了一个简单的协议来测量 C中时间过程ROS产生的水平。线虫使用H 2 DCFDA在正常和热或化学诱导的氧化应激条件下进行。使用该协议,我们确定了H 2 DCFDA浓度和测试蠕虫数量对DCF荧光信号的影响(图2)。

“”src
图1. ROS检测。A.通过细胞内ROS产生荧光DCF。 B.使用分子探针(H 2 DCFDA)在C中测量细胞内ROS。线虫。

“”src
图2.DCF荧光信号的变化取决于实验条件A.通过氧化应激表达gcs-1(启动子):: GFP(氧化应激标记)转基因。加热(30℃2小时)在活的线虫的肠中诱导了gcs-1(启动子):: GFP转基因的表达。 B.浓度 - 响应曲线以每个实验使用50个线虫的12.5,25,50和100μMH 2 DCFDA诱导的DCF荧光信号的平均值作图。 C.使用25μM的H 2 DCFDA评估取决于测试的线虫(10,20,50和100)数量的DCF荧光信号的变化。

关键字:活性氧族, 2',7'-二氯荧光素二乙酸盐, 秀丽隐杆线虫, 抗氧化剂

材料和试剂

  1. 1.5ml微量离心管(Corning,Axygen?,目录号:MCT-150-C)
  2. 锥形管,15ml(Corning,Falcon ,目录号:352096)
  3. 幻灯片(VWR,目录号:48300-025)
  4. 96孔微量培养板,黑色(SPL Life Sciences,目录号:30496)
  5. 60毫米培养皿(SPL Life Sciences,目录号:10060)
  6. 铂丝,0.2毫米(阿法埃莎,产品目录号:45093)
  7. 秀丽隐杆线虫野生型[N2]( Caenorhabditis 明尼苏达大学遗传学中心: https://cgc.umn.edu
  8. 大肠杆菌OP50菌株( Caenorhabditis Genetic Center)
  9. 蒸馏水
  10. 2',7'-二氯荧光素二乙酸酯(Sigma-Aldrich,目录号:D6883)
  11. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  12. 甲基紫精二氯化物水合物(Merck,目录号:856177)
  13. 胆固醇(Sigma-Aldrich,目录号:C8667)
  14. 乙醇(Sigma-Aldrich,目录号:E7023)
  15. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S5886)
  16. 细菌蛋白胨(BD,Bacto TM,目录号:211677)
  17. 琼脂(Sigma-Aldrich,目录号:A1296)
  18. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  19. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  20. 磷酸氢钾,二元酸(K 2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  21. 磷酸钾,一元(KH 2 PO 4)(Sigma-Aldrich,目录号:795488)
  22. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  23. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  24. 次氯酸钠(NaClO)(Sigma-Aldrich,目录号:425044)
  25. 5毫克/毫升胆固醇(见食谱)
  26. 线虫生长培养基(NGM)琼脂平板(见食谱)
  27. M9缓冲液(见食谱)
  28. 漂白液(见食谱)

设备

  1. 摇动培养箱(台式摇床培养箱,VWR,型号:1570型)
  2. Micropipette(VWR国际)
  3. 稳定温度的培养箱(VWR SIGNATURE 20-cuft型号2020 B.O.D. -10°C至45°C低温培养箱)(VWR,Advanced Instruments,型号:2020型)
  4. 台式离心机(VWR,型号:VWR Mini离心机C1413V)
  5. 解剖立体显微镜(具有复消色差光学徕卡S8 APO的立体显微镜)(Leica Microsystems,型号:Leica S8 APO)
  6. 荧光光度计(Promega,型号:GloMax®-Multi Microplate Multimode Reader)
  7. 涡旋混合器(标准重型涡旋混合器,VWR,目录号:97043-562)

  8. 高压灭菌器(Hanshin Medical,型号:HS-2321SD)

软件

  1. Microsoft Office 2017 Excel(Microsoft Corporation,Redmond,USA)
  2. IBM SPSS statistics 24(IBM Corporation,纽约,美国)

程序

  1. 在正常培养条件下测量线虫的细胞内ROS
    1. 准备一个同步的线虫种群
      1. 用1ml M9缓冲液洗涤含有完全成虫和鸡蛋的平板。
      2. 将线虫悬液转移到微量离心管中。
      3. 以3,884 emg(rcf; 8,500rpm)旋转1分钟并除去上清液。
      4. 加入200μl蒸馏水(D.W.)和800μl漂白溶液。
      5. 等待超过90%的线虫溶解并释放几个胚胎[请参阅Bianchi和Driscoll(2006)第3.2节)。
        注意:3分钟后每30秒检查一次溶解。以最大速度涡旋约2-3秒,有助于漂白剂溶解。
      6. 以3,884 emg(rcf; 8,500rpm)旋转1分钟并除去上清液。
        注意:小心不要打扰颗粒。
      7. 用M9缓冲液(各1ml)洗涤三次:以137gxg(rcf; 1,000rpm)离心1分钟并除去上清液。
      8. 用1ml M9缓冲液将胚胎转移到含有1ml M9(总共2ml)的锥形管中。

      9. 在20°C孵育过夜,摇动30转,让L1幼虫孵化。
    2. 计数动物和线虫解决方案的准备
      1. 重新悬浮幼虫沉淀,并将10微升悬浮液铺在载玻片上。这样做一式三份。

      2. 使用解剖立体显微镜计算线虫的数量,并计算每个液滴中L1幼虫的平均数量。
      3. 添加适量的M9缓冲液以稀释幼虫悬浮液。
      4. 轻轻摇动并重新计数。
      5. 重复步骤A2b-A2d,直到线虫数量达到每10μl约50个。
    3. 制备H 2 DCFDA反应溶液
      1. 通过将24.365mg H 2 DCFDA溶解在1ml细胞培养级别的DMSO中来制备浓缩储液(50mM)。
      2. 分成100微升等分试样在黑色的离心管中,并储存在-20°C(稳定至少3个月)。
      3. 根据需要用M9缓冲液将原液稀释至50μM(需要的体积=测试孔的数量x 50μl)。
        注意:工作解决方案应在使用前尽快准备。
    4. 使用荧光光度计测定细胞内ROS水平
      1. 打开荧光光度计并设置印版设置。

      2. 加入40μlM9缓冲液到黑色96孔板的孔中。

      3. 每孔加入10μl线虫溶液(步骤A2中制备)
      4. 加入50μlH 2 DCFDA反应溶液至终浓度为25μM。
      5. 通过加入50μl的M9和50μl的H 2 DCFDA反应溶液来制备空白孔。
      6. 立即将平板插入荧光光度计,轻轻摇动30秒。

      7. 在激发波长为490纳米和发射波长为510-570纳米时量化荧光强度
      8. 每小时测量荧光信号(最长6小时),并通过USB端口输出原始数据(见图2A)。
    注意:所有步骤都应在RT完成。

  2. 在氧化应激条件下测量线虫的细胞内ROS
    1. 百草枯诱导的氧化应激
      1. 在D.W.中溶解甲基紫精二氯化物水合物(百草枯)粉末制成50 mM百草枯溶液。
      2. 按步骤A1所述准备被捕的L1幼虫。
      3. 向9份含L1的M9缓冲液(1:10稀释)中加入1份50mM百草枯,并在摇动培养箱中以30rpm振荡在20℃孵育2小时。
      4. 用M9缓冲液(各1ml)洗涤三次:以137gxg(rcf; 1,000rpm)离心1分钟并除去上清液。
      5. 按照步骤A2所述准备线虫解决方案。
      6. 按照步骤A3和A4所述量化细胞内ROS水平。
    2. 热诱导的氧化应激
      1. 按步骤A1和A2所述准备被捕的L1幼虫和线虫溶液。

      2. 在30°C孵育线虫2小时,在摇动培养箱中摇动30 rpm。
      3. 如步骤A3和A4中所述量化细胞内ROS水平。
        注意:在整个测量过程中,将荧光光度计的温度保持在30°C。

数据分析

  1. 对于每种情况,使用至少20只动物来获得准确的结果。

  2. 每个实验至少应执行三次独立时间。
  3. 将空白扣除的原始数据(DCF荧光水平)归一化为野生型(或未处理)线虫的数据,并且使用Microsoft Office 2017 Excel软件以平均值±标准差表示。
  4. Tukey事后检验的单因素方差分析使用IBM SPSS统计数据24进行,以分析组间差异的统计显着性。

笔记

  1. 与许多其他荧光探针一样,H 2 DCFDA具有光敏性,因此处理时对光的最小曝光量至关重要。
  2. 当使用H 2 C DCFDA测量细胞内ROS时,应使用整体线虫代替裂解线虫。线虫。裂解导致外表皮和内膜的破坏,随后释放细胞内金属离子如铁。由于游离铁可以通过Fenton反应自身产生ROS,与线虫中的测量相比,裂解线虫中的ROS水平可能被高估。
  3. 由于H 2 DCFDA容易扩散到细胞中直到达到平衡,因此可以检测到增加的基础荧光信号的时间过程。在动物死亡的时间点,逐渐增加的趋势转变为急剧上升的趋势。因此,在开始实验后的4或6小时内进行测量是合适的。
  4. 成人线虫(年龄同步)也可用于测试化合物在氧化应激条件下的抗氧化能力,稍作修改。建议使用少量的线虫(10-20蠕虫/孔)。此外,增加百草枯浓度需要在成人线虫中诱导明显的氧化应激(超过20 mM,最终浓度)。但是,对于老化的线虫应该使用相对较低浓度的百草枯。

食谱

  1. 5毫克/毫升胆固醇
    将5毫克胆固醇粉末溶于1毫升100%乙醇中,并在4℃下储存
  2. 线虫生长培养基(NGM)琼脂平板(每L)
    3克的NaCl
    2.5克蛋白胨
    17克琼脂
    1毫升胆固醇溶液(5毫克/毫升)
    1毫升CaCl 2(1M)
    1毫升MgSO 4(1M)
    25毫升磷酸钾(1M,pH 6.0)

    975毫升蒸馏水 高压灭菌器并冷却至50°C
    将10毫升NGM琼脂倒入60毫米培养皿中并让其硬化1小时
  3. M9缓冲区
    3克KH 2 PO 4 4/2 6克Na 2 HPO 4 4 5克的NaCl
    1毫升MgSO 4(1M)
    用蒸馏水补充1升
    高压灭菌器
  4. 漂白解决方案
    6.25毫升4M NaOH
    2.5毫升的NaOCl
    50毫升的M9缓冲液

    使用前新鲜准备

致谢

这项工作得到了韩国国家研究基金会(NRF-2017R1C1B5015695)对DSC,Brody Brothers Endowment Grant(21602-664261),NIH(1R15GM112174-01A1),NSF(MCB1714264)向M-H.L的支持。 Caenorhabditis遗传中心(CGC)由美国国立卫生研究院 - 研究基础设施项目办公室(P40 OD010440)提供支持。该协议已由Yoon et al。改编,2017年。作者声明不存在相互竞争的利益。

参考

  1. Bianchi,L.和Driscoll,M。(2006)。 胚胎培养C。 elegans 用于电生理和药理学分析。 WormBook 1-15。
  2. Labuschagne,C.F。和Brenkman,A.B.(2013)。 当前在线虫体内定量ROS和氧化损伤的方法和其他模型衰老有机体 衰老修复 12(4):918-930。
  3. Miranda-Vizuete,A.和Veal,E.A。(2017)。 秀丽隐杆线虫(Caenorhabditis elegans)作为理解生理和疾病中ROS功能的模型。 Redox Biol 11:708-714。
  4. Yoon,DS,Choi,Y.,Cha,DS,Zhang,P.,Choi,SM,Alfhili,MA,Polli,JR,Pendergrass,D.,Taki,FA,Kapalavavi,B.,Pan,X.,Zhang ,B.,Blackwell,TK,Lee,JW和Lee,MH(2017)。 三氯生破坏SKN-1 / Nrf2介导的氧化应激反应。线虫和人类间充质干细胞。 Sci Rep 7(1):12592。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yoon, D. S., Lee, M. and Cha, D. S. (2018). Measurement of Intracellular ROS in Caenorhabditis elegans Using 2’,7’-Dichlorodihydrofluorescein Diacetate. Bio-protocol 8(6): e2774. DOI: 10.21769/BioProtoc.2774.
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