秀丽隐杆线虫中细胞内ATP水平的评估

Konstantinos Palikaras; Nektarios  Tavernarakis

doi:10.21769/BioProtoc.2048

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Peer-reviewed

Intracellular Assessment of ATP Levels in Caenorhabditis elegans

秀丽隐杆线虫中细胞内ATP水平的评估

Konstantinos Palikaras

Nektarios Tavernarakis email

发布: 2016年12月05日第6卷第23期 DOI: 10.21769/BioProtoc.2048 浏览次数: 10942

评审: Jyotiska ChaudhuriAnonymous reviewer(s)

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Abstract

Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans.

Keywords: Ageing (衰老)

ATP (ATP)

Caenorhabditis elegans (秀丽隐杆线虫)

Energy homeostasis (能量稳态)

Luciferase (荧光素酶)

Metabolism (代谢)

Mitochondria (线粒体)

Background

Mitochondria-derived ATP plays a crucial role in a variety of cellular and metabolic processes. Therefore, several methods have been developed to measure the levels of this important metabolite (Drew and Leeuwenburgh, 2003; Khan, 2003; Lagido et al., 2001; Vives-Bauza et al., 2007). In 1947, McElroy proposed the use of firefly bioluminescence to determine intracellular ATP levels, when he uncovered the essential role of ATP in light production (McElroy, 1947). The development of cloning and recombinant protein technology facilitated the development of the firefly luciferase assay to determine ATP levels (de Wet et al., 1985). Firefly luciferase, a monomeric 61 kD enzyme, catalyzes the oxidation of luciferin, which emits light at 560 nm. When ATP is the limiting factor of the enzymatic reaction, the intensity of light is proportional to the concentration of ATP. Thus, the use of a luminometer permits the detection of light intensity and subsequently the determination of ATP levels. In this protocol, we describe a method for ATP quantification using a bioluminescence approach in C. elegans.

Materials and Reagents

1.5 ml tube
Greiner Petri dishes (60 x 15 mm) (Greiner Bio One, catalog number: 628161 )
Toothpick
L4 larvae
C. elegans strains (wild type [N2] and pink-1[tm1779])
Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
Liquid nitrogen
Lyophilized ATP (provided with ATP bioluminescence assay kit CLS II) (Roche Diagnostics, catalog number: 11699695001 )
Distilled water
Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 23225 )
Glue
70% of EtOH
ATP bioluminescence assay kit CLS II (Roche Diagnostics, catalog number: 11699695001 )
Sodium chloride (NaCl) (EMD Millipore, catalog number: 1064041000 )
Bacto^TM peptone (BD, catalog number: 211677 )
Streptomycin (Sigma-Aldrich, catalog number: S-6501 )
Agar (Sigma-Aldrich, catalog number: 05040 )
Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C-5080 )
Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M-7506 )
Nystatin stock solution (Sigma-Aldrich, catalog number: N-3503 )
Potassium dihydrogen phosphate (KH₂PO₄) (EMD Millipore, catalog number: 1048731000 )
Na₂HPO₄ (EMD Millipore, catalog number: 1065860500 )
K₂HPO₄
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P-5405 )
Phosphate buffer (1 M; sterile, see Recipes)
Nematode growth medium (NGM) agar plates (see Recipes)
M9 buffer (see Recipes)

Equipment

Dissecting stereomicroscope (Olympus, model: SMZ645 )
Incubators for stable temperature (AQUA^®LYTIC incubator 20 °C)
TD-20/20 luminometer (Turner Designs, model: 2020-000 )
Tabletop centrifuge (Eppendorf, model: 5424 )
Freezers (Siemens, -20 °C; So-Low Environmental Equipment, model: So-Low Ultra C85-22 horizontal -80 °C freezer)
Heat plate
Hot pot

Software

Microsoft Office 2011 Excel (Microsoft Corporation, Redmond, USA)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Palikaras, K. and Tavernarakis, N. (2016). Intracellular Assessment of ATP Levels in Caenorhabditis elegans. Bio-protocol 6(23): e2048. DOI: 10.21769/BioProtoc.2048.