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Peer-reviewed

Measurement of ROS in Caenorhabditis elegans Using a Reduced Form of Fluorescein

使用还原型荧光素测定秀丽隐杆线虫中的ROS

Shaarika Sarasija Shaarika Sarasija
KN Kenneth R. Norman

发布: 2018年04月05日第8卷第7期 DOI: 10.21769/BioProtoc.2800 浏览次数: 6714

评审: Tugsan TezilJuan Facundo Rodriguez AyalaAnonymous reviewer(s)

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Abstract

Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer’s disease. Oxidative stress is a result of a disruption of the equilibrium between antioxidants and oxidants, in favor of oxidants. Since mitochondria are major sites of production and reduction of reactive oxygen species (ROS), measurement of ROS levels can help us determine if mitochondrial functional integrity has been compromised. In this protocol, we describe a method to measure the level of ROS in the nematode Caenorhabditis elegans, using chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA).

Keywords: C. elegans (秀丽隐杆线虫) search ROS (ROS) search Mitochondria (线粒体) search DCF (DCF) search Oxidative stress (氧化应激) search CM-H2DCFDA (CM-H2DCFDA) search

Background

The life cycle of ROS is closely associated with the mitochondria. Superoxides are produced as a result of the inevitable electron leak at complex I and complex III from the electron transport chain in the mitochondria. This superoxide is then dismutated to hydrogen peroxide in the mitochondrial matrix and mitochondrial intermembrane space. The superoxide and hydrogen peroxide generated via these processes are considered to be ROS, with the mitochondria being responsible for 90% of endogenous ROS (Balaban et al., 2005). Therefore, measuring ROS levels will provide insight into the status of mitochondrial health. Here, we provide a protocol to determine the levels of ROS in the nematode C. elegans using CM-H2DCFDA, a cell-permeant chloromethyl derivative of a reduced form of fluorescein. Once inside the cell, the CM-H2DCFDA is cleaved by intracellular esterases and the resulting nonfluorescent H2DCF, upon oxidation by ROS, gets converted into the highly fluorescent 2’,7’-dichlorofluorescein (DCF). Imaging and measurement of fluorescence intensity of H2DCFDA in live C. elegans can be confounded by the presence of autofluorescence from the animal’s intestine. Our protocol utilizes worm lysates to measure ROS levels, thereby bypassing this issue (Sarasija and Norman, 2015).

Materials and Reagents

  1. 100 mm, 60 mm Petri dishes (Kord-Valmark Labware Products, catalog numbers: 2900 , 2901 )
  2. 15-ml centrifuge tubes (Globe Scientific, catalog number: 6285 )
  3. 22 x 22 mm coverslip (Globe Scientific, catalog number: 1404-10 )
  4. Glass Pasteur pipettes (Krackeler Scientific, catalog number: 6-72050-900 )
  5. Vacuum filtration (GE Healthcare, Whatman, catalog number: 6722-5001 )
  6. 1.5 ml Micro Centrifuge tube (CELLTREAT Scientific, catalog number: 229443 )
  7. 50 ml conical tubes (Corning, catalog number: 430829 )
  8. 15 ml conical tubes (Corning, catalog number: 430791 )
  9. C. elegans strains and OP50 (Caenorhabditis Genetics Center (CGC), University of Minnesota)
  10. Deionized water (dH2O)
  11. BCA protein assay (Pierce, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23227 )
  12. Chloro methyl-2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C6827 )
  13. Polybead Polystyrene 0.10 μm microspheres (Polysciences, catalog number: 00876-15 )
  14. Agarose (RPI, catalog number: A20090-500.0 )
  15. Clear nail polish (generic)
  16. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-10 )
  17. Agar (Fisher Scientific, catalog number: BP1423-2 )
  18. Bacto peptone (BD, BactoTM, catalog number: 211677 )
  19. Calcium chloride dihydrate (CaCl2·2H2O) (Fisher Scientific, catalog number: C79-500 )
  20. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Fisher Scientific, catalog number: BP213-1 )
  21. Potassium phosphate dibasic (K2HPO4) (Fisher Scientific, catalog number: BP363-1 )
  22. Cholesterol (Fisher Scientific, catalog number: C314-500 )
  23. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285-500 )
  24. Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366-1 )
  25. 5-Fluoro-2’-deoxyuridine (FUDR) (bioWORLD, catalog number: 40690016-1 )
  26. Bacto tryptone (BD, BactoTM, catalog number: 211705 )
  27. Bacto yeast extract (BD, BactoTM, catalog number: 212750 )
  28. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: BP359-500 )
  29. Sodium phosphate dibasic anhydrous (Na2HPO4) (Fisher Scientific, catalog number: BP332-1 )
  30. Bleach (generic, plain)
  31. Hydrogen chloride (HCl) (Fisher Scientific, catalog number: A144-500 )
  32. Standard Nematode Growth Media (NGM) plates (see Recipes)
  33. Sterile solutions (see Recipes)
  34. 5-Fluoro-2’-deoxyuridine containing NGM plates (see Recipes)
  35. Sterile stocks for NGM (see Recipes)
    1. 1 M CaCl2
    2. 1 M MgSO4
    3. 1 M K2HPO4
    4. 1 M KH2PO4
    5. 1 M KPO4
  36. LB medium (see Recipes)
  37. M9 buffer (see Recipes)
  38. Bleach solution (see Recipes)
  39. 10 N NaOH (see Recipes)
  40. 10x PBS (see Recipes)

Equipment

  1. Single channel pipettes (Rainin, models: PR-10 , PR-20 , PR-200 , PR-1000 )
  2. Finnpipette II Multichannel pipettes (Fisher Scientific, model: FisherbrandTM FinnpipetteTM II, catalog number: 21377830 )
  3. Fisher Scientific Sonic Dismembrator with microtip probe (Fisher Scientific, model: Sonic Dismembrator Model 100 )
  4. Thermo Electron Corporation IEC Centra CL2 Centrifuge (Thermo Fischer Scientific, Thermo ScientificTM, model: IEC Centra CL2 )
  5. Eppendorf 5415D Centrifuge (Eppendorf, model: 5415 D )
  6. Castle Steam Sterilizer Autoclave (Getinge, model: 433/533HC-E )
  7. Zeiss SteREO Discovery. V8 microscope with SCHOTT Ace® I light source (ZEISS, model: SteREO Discovery.V8 )
  8. Molecular Devices FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, model: FlexStation® 3 )
  9. PYREX® Griffin beakers (Corning, catalog number: 1000-PACK )
  10. PYREX® Reusable Media Storage Bottles (Fisher Scientific)

Software

  1. SoftMax® Pro 6 Software (Molecular Devices)
  2. Microsoft Office 2011 Excel (Microsoft Corporation, Redmond, USA)
  3. GraphPad Prism 5 software package (GraphPad Software Inc., San Diego, USA)

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Sarasija, S. and NORMAN, K. R. (2018). Measurement of ROS in Caenorhabditis elegans Using a Reduced Form of Fluorescein. Bio-protocol 8(7): e2800. DOI: 10.21769/BioProtoc.2800.

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分类

神经科学 > 神经系统疾病 > 动物模型

细胞生物学 > 细胞新陈代谢 > 其它化合物

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