秀丽隐杆线虫中耗氧速率测定

Konstantinos Palikaras; Nektarios  Tavernarakis

doi:10.21769/BioProtoc.2049

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Peer-reviewed

Measuring Oxygen Consumption Rate in Caenorhabditis elegans

秀丽隐杆线虫中耗氧速率测定

Konstantinos Palikaras

Nektarios Tavernarakis email

发布: 2016年12月05日第6卷第23期 DOI: 10.21769/BioProtoc.2049 浏览次数: 10457

评审: Jyotiska ChaudhuriJian ChenAnonymous reviewer(s)

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Abstract

The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras et al., 2015; Ryu et al., 2016) or investigate factors mediating the switch from oxidative phosphorylation to aerobic glycolysis (Chen et al., 2015; Vander Heiden et al., 2009). In this protocol, we describe a method for the determination of oxygen consumption rates in the nematode Caenorhabditis elegans.

Keywords: Ageing (衰老)

Caenorhabditis elegans (秀丽隐杆线虫)

Background

Recent evidence underlines mitochondrial function as a potential contributor in the maintenance of organismal homeostasis and viability (Vafai and Mootha, 2012). Cellular oxygen consumption is highly recognized as a fundamental indicator of mitochondrial function, reflecting reactive oxygen species (ROS) production and metabolic activity of the cell. Therefore, several methods have been developed to measure oxygen consumption rates in cells or entire organisms (Dranka et al., 2011; Li and Graham, 2012; Luz et al., 2015; Perry et al., 2013). These approaches provided insight into the pivotal roles of mitochondria in disease progression and pathogenesis (Scheibye-Knudsen et al., 2015). In this protocol, we describe a method for the determination of oxygen consumption rates in the nematode C. elegans by using a Clark-type polarographic oxygen sensor electrode (Hansatech, King’s Lynn, England).

Materials and Reagents

Consumables
1. Commercial cigarette paper
2. Polytetrafluorethylene (PTFE) membrane (provided by Hansatech, King’s Lynn, England)
3. 15 ml tube (SARSTEDT, catalog number: 62.554.016 )
4. 1.5 ml tube (Sigma-Aldrich, catalog number: Z606340 )
5. Paper towel
6. Greiner Petri dishes (60 x 15 mm) (Greiner Bio One, catalog number: 628161 )
7. Maintenance kit (Hansatech, King’s Lynn, England)
Biologicalreagents
1. C. elegans strains (wild type [N2] and dct-1[tm376])
2. Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
Chemical reagents
1. Distilled water
2. Nitrogen gas provided in a tank
3. Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 23225 )
4. Potassium dihydrogen phosphate (KH₂PO₄) (EMD Millipore, catalog number: 1048731000 )
5. K₂HPO₄
6. Sodium chloride (NaCl) (EMD Millipore, catalog number: 1064041000 )
7. Bacto^TM peptone (BD, Bacto^TM, catalog number: 211677 )
8. Streptomycin (Sigma-Aldrich, catalog number: S-6501 )
9. Agar (Sigma-Aldrich, catalog number: 05040 )
10. Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
11. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C-5080 )
12. Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M-7506 )
13. Nystatin stock solution (Sigma-Aldrich, catalog number: N-3503 )
14. Na₂HPO₄ (EMD Millipore, catalog number: 1065860500 )
15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P-5405 )
16. Potassium chloride (KCl) buffer (see Recipes)
17. Nematode growth medium (NGM) agar plates (see Recipes)
18. M9 buffer (sterile; see Recipes)
19. Phosphate buffer (sterile; see Recipes)

Equipment

Dissecting stereomicroscope (Olympus, model: SMZ645 )
Incubators for stable temperature (AQUA^®LYTIC incubator 20 °C)
DW1/AD Clark-type polarographic oxygen sensor (Hansatech Instruments, model: Oxygraph Plus System )
Water bath at 20 °C
Tabletop centrifuge (Eppendorf, model: 5424 )
Sonicator (Sonics & Material, model: VC 130PB )

Software

Oxygraph Plus software (Hansatech, King’s Lynn, England)
Microsoft Office 2011 Excel

Procedure

English

中文翻译

文章信息

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如何引用

Palikaras, K. and Tavernarakis, N. (2016). Measuring Oxygen Consumption Rate in Caenorhabditis elegans. Bio-protocol 6(23): e2049. DOI: 10.21769/BioProtoc.2049.