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Measuring Oxygen Consumption Rate in Caenorhabditis elegans
秀丽隐杆线虫中耗氧速率测定   

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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), Metabolism(代谢), Mitochondria(线粒体), Oxygen sensor(氧传感器), ROS(ROS)

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

  1. 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)

  2. Biological reagents

    1. C. elegans strains (wild type [N2] and dct-1[tm376])
    2. Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)

  3. Chemical reagents
    1. Distilled water
    2. Nitrogen gas provided in a tank
    3. PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, 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. BactoTM peptone (BD, BactoTM, 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

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

Software

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

Procedure

  1. Growth and synchronization of nematode population
    1. Use a dissecting stereomicroscope to select L4 larvae of each strain. Place 4-6 L4 larvae on a freshly Escherichia coli (OP50) seeded NGM plate (Figures 1A-1C). Use at least three plates for each nematode strain.


      Figure 1. Caenorhabditis elegans in the laboratory. A. E. coli (OP50) seeded NGM plates on the base of a dissecting stereomicroscope. Bacterial lawn is visible on the surface of the agar. B. A mix population of nematodes observed through the dissecting stereomicroscope. Nematodes leave tracks on the plate surface indicating their movements on the bacterial lawn. Scale bar = 1 mm. C. During development worms increase in size throughout four larval stages (L1, L2, L3 and L4). L4 hermaphrodite nematodes can be distinguished by the developing vulva (red arrowhead), which is a clear half circle in the center of the ventral side. Scale bar = 0.5 mm.

    2. Incubate the worms at the standard temperature of 20 °C.
    3. Four days later the plates contain mixed nematode population (Figure 1B).
    4. Synchronize nematodes by picking L4 larvae of each strain under a dissecting stereomicroscope and transfer them onto separate plates.
    5. Add 20-25 L4 larvae per plate. For each experimental condition, use at least five plates.

  2. Set up the electrode disc and the electrode chamber
    1. Read the manual to assemble properly the electrode chamber according to the manufacturers’ instructions.
    2. Cut a 1.5 cm2 paper spacer (cigarette paper) and a similar size piece of polytetrafluorethylene (PTFE) membrane (Figure 2A; Video 1).


      Figure 2. Oxygen consumption rate in C. elegans. A. Spare parts of oxygraph apparatus: 1. Oxygraph electrode control unit, 2. Oxygraph chamber (top plate, water jacket, borosilicate glass reaction vessel and base ring, 3. Polytetrafluorethylene (PTFE) membrane, 4. Commercial cigarette papers, 5. Membrane applicator, 6. Electrode disc, 7. Small and large O-rings, 8. Standard plunger assembly, 9. Oxygraph Plus software, 10. Electrode maintenance kit (cotton buds and rapid Hansatech polishing paste). B. Slopes depict oxygen consumption rate upon measurements of wild type (2, 4, 6) and dct-1(tm376) (1, 3, 5) animals. C. Oxygen consumption rates normalized to total protein content. DCT-1 depleted animals display increased oxygen consumption levels (*** P < 0.001; unpaired t-test). Error bars denote SEM.

      Video 1. Preparation of electrode disc and chamber during oxygen consumption measurement

    3. Place a small droplet of potassium chloride (KCl) buffer on the top of the dome of the electrode disc (Video 1).
    4. Place the cigarette paper on the center of the dome and cover it with the similar size piece of PTFE membrane (Figure 2A; Video 1).
    5. Place the small O-ring over the dome and then place the large O-ring around (Figure 2A; Video 1). If the large O-ring is not placed, when the disc is installed in the electrode chamber, the measurements will be affected by ambient air due to impaired sealing (see Note 1; Video 1).
    6. Once the electrode disc has been successfully prepared, check the response of the disc prior to any experiment. Connect the electrode disc to the control unit.
    7. Open the software and start recording. A few minutes later the signal will be stabilized and would be around 2,000 mV in air.
    8. Test the electrode disc by breathing on the electrode. A steep drop of the signal should be observed due to decreased oxygen levels. Then, the signal should be returned to the original levels as the ambient oxygen equilibrates around the electrode disc.
    9. If the signal does not respond, it may be caused by an inadequate electrode preparation. Then, disassemble the electrode disc, clean the electrode and repeat the procedure described above (steps B1-B8).

  3. Liquid phase calibration of the electrode disc
    1. Install the prepared electrode disc into the electrode chamber (Figure 2A, Video 1).
    2. Place and connect the electrode chamber on to the rear of the control unit (Figure 2A).
    3. Place 2 ml sterilized distilled water into the reaction vessel of the chamber.
    4. Water should be equilibrated to the assay temperature before calibration process. Set up the water bath at desired temperature and connect it with the electrode chamber to maintain stable temperature during experiments (see Note 2).
    5. Open Oxygraph Plus software and set up the appropriate temperature and atmospheric pressure.
    6. Turn on stirrer to provide smooth stirring of the sample avoiding bubbles generation, which could cause noisy signals.
    7. Wait until a plateau of the signal has been reached.
    8. Establish zero oxygen conditions. Bubble nitrogen gas into the reaction vessel of the chamber to get rid all the oxygen out of the sample. Wait until the signal has reached a plateau.
    9. Add 1 ml M9 buffer into the reaction vessel of the chamber and wait until system equilibration.

  4. Assess oxygen consumption rate of the samples
    1. Enable stirring throughout the duration of the experimental process.
    2. Wash off the NGM plates using M9 buffer and collect nematodes in a 15 ml tube.
    3. Let the animals to settle with gravity for few minutes. Use adult worms. Remove most of the supernatant, which contains bacteria, eggs and larvae (L1, L2, L3 and L4). Repeat this step two more times. After the last wash, reduce the volume to ~1.5 ml.
    4. Remove M9 buffer from the reaction vessel of the chamber (see Note 3).
    5. Add 1 ml nematode suspension into the reaction vessel of the chamber.
    6. Monitor oxygen consumption for 5-10 min.
    7. Prepare the next sample. Wash off other plates containing different strains or treated animals. Repeat steps D3-D5.
    8. Recover the animals thoroughly from the reaction vessel and place them in a 1.5 ml tube (see Note 3).
    9. Place the tube on ice. The tubes should be kept on ice until protein determination.
    10. Measure the next sample. Repeat steps D5-D9 (see Note 4).
    11. Save the recorded data from Oxygraph Plus software (Figure 2B).
    12. Collect the samples from ice and proceed to worm lysis and protein determination.
    13. Insert sonicator tip into sample and sonicate each sample using ten (10) pulses at 70% power each time. Immediately place the samples on ice (see Note 5).
    14. Spin down samples at 14,800 x g for 10 min at 4 °C.
    15. Transfer supernatants (solubilized proteins) into new 1.5 ml tubes and measure protein concentration using a standard protein kit, such as PierceTM BCA Protein Assay Kit, following the instructions of the manufacturer (Estimated protein concentration range: 0.05-0.9 μg).
    16. Open and review the data (obtained during step D11) in Microsoft Office 2011 Excel (Figure 2B).
    17. Divide the rate of the negative slope (oxygen consumption; obtained during step D6) with the total protein amount (nmol/min/ml/mg) in Microsoft Office 2011 Excel software (Figure 2C).
    18. Subject the data to further and more advanced statistical analysis (Figure 2C).

  5. Electrode maintenance and storage
    1. Clean the electrode disc after use.
    2. Use the small cotton bud and the polishing paste from the maintenance kit.
    3. Grip gently and remove the brown/black deposition of silver chloride and oxidized salt (KCl) that normally generated on silver electrode (see Note 6). Repeat polishing until all brown/black deposits on the electrode surface are removed.
    4. Gently polish the center of the electrode dome avoiding scratching.
    5. Rinse the electrode disc with distilled water to remove all traces of the polishing paste (see Note 7).
    6. Dry the electrode disc by using a paper towel.
    7. Store the electrode disc in an air-tight vessel.

Data analysis

  1. For each strain or condition, use at least 100 animals to obtain more accurate results.
  2. For each experiment, the same number of nematodes should be examined for each strain and condition.
  3. Each assay should be repeated at least three (3) times.
  4. Use the Student’s t-test with a significance cut-off level of P < 0.05 for comparisons between two groups.
  5. Use the one-factor (ANOVA) variance analysis and correct by the post hoc Bonferroni test for multiple comparisons.

Notes

  1. Do not over-tighten the large O-ring by screwing too strong because it could cause drifting signals during measurements.
  2. Oxygen electrode discs are very sensitive to temperature fluctuations affecting oxygen levels. Maintain experimental temperature stable throughout the duration of the experiment. Equilibrate the electrode disc to the assay temperature before taking place any calibration and/or measurement.
  3. Be careful not to destroy the membrane of the electrode disc during buffer removal.
  4. Perform three independent measurements per strain or condition.
  5. During sonication, samples should be kept cold to avoid protein degradation. Incubate samples constantly on ice during sonication.
  6. The electrode disc should never be left to dry out with KCl in place. Crystallization of KCl remnants could oxidized and destroy the electrode. Maintaining the disc to a high standard is extremely important.
  7. Do not wet the electrical connector of the disc during maintenance process.

Recipes

  1. Phosphate buffer (1 M)
    1. For 1 L, dissolve 102.2 g KH2PO4 and 57.06 g K2HPO4 in distilled water and fill up to 1 L. This is a 1 M solution, pH 6.0
    2. Autoclave and store at room temperature
  2. Nematode growth medium (NGM) agar plates
    1. Mix 3 g NaCl, 2.5 g BactoTM peptone, 0.2 g streptomycin, 17 g agar and add 900 ml distilled water. Autoclave
    2. Let cool to 55-60 °C
    3. Add 1 ml cholesterol stock solution, 1 ml 1 M CaCl2, 1 ml 1 M MgSO4 (sterile), 1 ml nystatin stock solution, 25 ml sterile 1 M phosphate buffer, pH 6.0, and distilled sterile water up to 1 L
    4. Pour about 8 ml medium per Petri dish and leave to solidify
    5. Keep the plates at 4 °C until used
  3. M9 buffer
    1. Dissolve 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl in 1 L distilled water. Autoclave
    2. Let cool and add 1 ml 1 M MgSO4 (sterile)
    3. Store M9 buffer at 4 °C
  4. Potassium chloride (KCl) buffer
    1. Dissolve 17.5 g KCl in 100 ml distilled water
    2. Store KCl buffer at 4 °C

Acknowledgments

This work was funded by grants from the European Research Council (ERC), the European Commission 7th Framework Programme and Bodossaki Foundation Postdoctoral Research Fellowship. The protocol has been adapted from Palikaras et al. (2015) Nature 521, 525-528.

References

  1. Artal-Sanz, M. and Tavernarakis, N. (2009). Prohibitin couples diapause signalling to mitochondrial metabolism during ageing in C. elegans. Nature 461(7265): 793-797.
  2. Chen, Z., Wang, Z., Guo, W., Zhang, Z., Zhao, F., Zhao, Y., Jia, D., Ding, J., Wang, H., Yao, M. and He, X. (2015). TRIM35 Interacts with pyruvate kinase isoform M2 to suppress the Warburg effect and tumorigenicity in hepatocellular carcinoma. Oncogene 34(30): 3946-3956.
  3. Dranka, B. P., Benavides, G. A., Diers, A. R., Giordano, S., Zelickson, B. R., Reily, C., Zou, L., Chatham, J. C., Hill, B. G., Zhang, J., Landar, A. and Darley-Usmar, V. M. (2011). Assessing bioenergetic function in response to oxidative stress by metabolic profiling. Free Radic Biol Med 51(9): 1621-1635.
  4. Li, Z., and Graham, B. H. (2012). Measurement of mitochondrial oxygen consumption using a Clark electrode. Methods Mol Biol 837: 63-72.
  5. Luz, A. L., Rooney, J. P., Kubik, L. L., Gonzalez, C. P., Song, D. H. and Meyer, J. N. (2015). Mitochondrial morphology and fundamental parameters of the mitochondrial respiratory chain are altered in Caenorhabditis elegans strains deficient in mitochondrial dynamics and homeostasis processes. PLoS One 10(6); e0130940.
  6. Palikaras, K., Lionaki, E. and Tavernarakis, N. (2015). Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans. Nature 521(7553): 525-528.
  7. Perry, C. G., Kane, D. A., Lanza, I. R. and Neufer, P. D. (2013). Methods for assessing mitochondrial function in diabetes. Diabetes 62(4): 1041-1053.
  8. Ryu, D., Mouchiroud, L., Andreux, P. A., Katsyuba, E., Moullan, N., Nicolet-Dit-Felix, A. A., Williams, E. G., Jha, P., Lo Sasso, G., Huzard, D., Aebischer, P., Sandi, C., Rinsch, C. and Auwerx, J. (2016). Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents. Nat Med 22(8): 879-888.
  9. Scheibye-Knudsen, M., Fang, E. F., Croteau, D. L., Wilson, D. M., 3rd, and Bohr, V. A. (2015). Protecting the mitochondrial powerhouse. Trends in cell biology 25(3): 158-170.
  10. Vafai, S. B. and Mootha, V. K. (2012). Mitochondrial disorders as windows into an ancient organelle. Nature 491(7424): 374-383.
  11. Vander Heiden, M. G., Cantley, L. C. and Thompson, C. B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324(5930): 1029-1033.

简介

氧消耗速率是指示在正常或代谢挑战条件下的寿命期间的细胞功能的重要标志物。它广泛用于研究线粒体功能(Artal-Sanz和Tavernarakis,2009; Palikaras等人,2015; Ryu等人,2016)或调查调节从氧化磷酸化转变为有氧糖酵解(Chen等人,2015; Vander Heiden等人,2009)。在该方案中,我们描述了用于确定线虫中线虫的氧消耗速率的方法。
关键字:老化,线虫最近的证据强调了线粒体功能作为维持生物体内平衡和生存能力的潜在贡献者(Vafai和Mootha) ,2012)。细胞氧消耗被高度认为是线粒体功能的基本指标,反映细胞的活性氧(ROS)产生和代谢活性。因此,已经开发了几种方法来测量细胞或整个生物体中的氧消耗速率(Dranka等人,2011; Li和Graham,2012; Luz等人, 2015; Perry et al 。,2013)。这些方法提供了对线粒体在疾病进展和发病机理中的关键作用的了解(Scheibye-Knudsen等人,2015)。在该方案中,我们描述了测定线虫中氧消耗速率的方法。 elegans ,使用Clark型极谱氧传感器电极(Hansatech,King's Lynn,England)。

关键字:衰老, 秀丽隐杆线虫, 代谢, 线粒体, 氧传感器, ROS

材料和试剂

  1. 消耗品
    1. 商业卷烟纸
    2. 聚四氟乙烯(PTFE)膜(由Hansatech,King's Lynn,England提供)
    3. 15ml管(STARSTEDT,目录号:62.554.016)
    4. 1.5ml管(Sigma-Aldrich,目录号:Z606340)
    5. 毛巾
    6. Greiner Petri dish(60×15mm)(Greiner Bio One,目录号:628161)
    7. 维护套件(Hansatech,King's Lynn,England)

  2. C。 elegans 菌株(野生型[N2]和 dct-1 [tm376] )
  3. 大肠杆菌 OP50菌株(获自Caenorhabditis Genetics Center)

  • 化学试剂
    1. 蒸馏水
    2. 罐中提供的氮气
    3. PierceBCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23225)
    4. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:1048731000)
    5. K 2 HPO 4
    6. 氯化钠(NaCl)(EMD Millipore,目录号:1064041000)
    7. Bacto TM 蛋白胨(BD,Bacto TM ,目录号:211677)
    8. 链霉素(Sigma-Aldrich,目录号:S-6501)
    9. 琼脂(Sigma-Aldrich,目录号:05040)
    10. 胆固醇储备溶液(SERVA电泳,目录号:17101.01)
    11. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C-5080)
    12. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M-7506)
    13. 制霉菌素储备液(Sigma-Aldrich,目录号:N-3503)

    14. (EMD Millipore,目录号:1065860500)
    15. 氯化钾(KCl)(Sigma-Aldrich,目录号:P-5405)
    16. 氯化钾(KCl)缓冲液(见配方)
    17. 线虫生长培养基(NGM)琼脂平板(见Recipes)
    18. M9缓冲液(无菌;参见配方)
    19. 磷酸盐缓冲液(无菌;参见配方)
  • 设备

    1. 解剖立体显微镜(Olympus,型号:SMZ645)
    2. 稳定温度的培养箱(AQUA LYTIC培养箱20°C)
    3. DW1/AD clark型极谱氧传感器(Hansatech Instruments,型号:Oxygraph Plus System)
    4. 20℃水浴
    5. 台式离心机(Eppendorf,型号:5424)
    6. 超声波仪(Sonics& Material,型号:VC 130PB)

    软件

    1. Oxygraph Plus软件(Hansatech,King's Lynn,England)
    2. Microsoft Office 2011 Excel

    程序

    1. 线虫群体的生长和同步
      1. 使用解剖立体显微镜选择每个菌株的L4幼虫。将4-6LL幼虫放在新鲜大肠杆菌(OP50)接种的NGM板上(图1A-1C)。每种线虫菌株至少使用三块板。


        图1.实验室中的Caenorhabditis elegans 。 A. 大肠杆菌(OP50)在解剖立体显微镜的基部上接种NGM平板。细菌草坪在琼脂的表面上可见。 B.通过解剖立体显微镜观察的线虫的混合群体。线虫在板表面上留下轨迹,指示它们在细菌草坪上的移动。比例尺= 1mm。 C.在发育期间,蠕虫在四个幼虫阶段(L1,L2,L3和L4)中的尺寸增加。 L4雌雄同体线虫可以通过发育的外阴(红色箭头)区分,其是在腹侧中心的清楚的半圆。比例尺= 0.5mm。

      2. 在20°C的标准温度下孵育蠕虫。
      3. 四天后,盘子含有混合的线虫群(图1B)
      4. 通过在解剖立体显微镜下挑取每个菌株的L4幼虫来同步线虫,并将它们转移到单独的平板上。
      5. 每板添加20-25LL幼虫。对于每个实验条件,至少使用五块板
    2. 设置电极盘和电极室
      1. 阅读手册,根据制造商的说明正确组装电极室
      2. 切割1.5cm的纸隔片(卷烟纸)和类似尺寸的聚四氟乙烯(PTFE)膜(图2A;视频1)。


        图2. C中的氧消耗率。 。A.氧测量仪器的备件:1.氧测量电极控制单元2.氧测量室(顶板,水套,硼硅酸盐玻璃反应容器和基环,3.聚四氟乙烯膜,4.商业香烟纸,5.膜施加器,6.电极盘,7.小和大O型环,8.标准柱塞组件,9.Oxygraph Plus软件,10.电极维护套件(棉花芽和快速Hansatech抛光糊)B.斜率描绘了野生型(2,4,6)和dct-1(tm376)(1,3,5)动物测量时的氧消耗速率C.氧消耗对于总蛋白含量标准化的速率,DCT-1耗尽的动物显示出增加的氧消耗水平(***

        <0.001>未配对的测试) 。

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      3. 将一小滴氯化钾(KCl)缓冲液放在电极圆盘的顶部(视频1)。
      4. 将卷烟纸放在圆顶的中心,并用类似尺寸的PTFE膜覆盖(图2A;视频1)。
      5. 将小O形圈放在圆顶上,然后放置大O形圈(图2A;视频1)。如果没有放置大O型圈,则当盘安装在电极室中时,由于密封受损,测量将受环境空气的影响(见注1;视频1)。
      6. 一旦电极盘已经成功准备好,在任何实验之前检查盘的响应。将电极盘连接到控制单元。
      7. 打开软件并开始录制。几分钟后,信号将稳定,并在空气中为约2,000mV。
      8. 通过呼吸电极测试电极盘。由于氧水平降低,应该观察到信号的急剧下降。然后,当环境氧气在电极盘周围平衡时,信号应该返回到原始水平
      9. 如果信号没有响应,则可能是由不适当的电极准备引起的。然后,拆开电极盘,清洁电极,并重复上述步骤(步骤B1-B8)。

    3. 电极盘的液相校准
      1. 将准备的电极盘安装到电极室(图2A,视频1)。
      2. 将电极室放置并连接到控制单元的后部(图2A)。
      3. 将2毫升无菌蒸馏水放入反应器的反应容器中
      4. 在校准过程之前,应将水平衡至测定温度。将水浴设置在所需温度,并将其与电极室连接,以在实验过程中保持稳定的温度(见注2)。
      5. 打开Oxygraph Plus软件,设置适当的温度和大气压力
      6. 打开搅拌器以平稳地搅拌样品,避免产生气泡,这可能会导致噪音信号
      7. 等待直到达到信号的平稳。
      8. 建立零氧条件。将氮气鼓入腔室的反应容器中以除去样品中的所有氧气。等待信号达到平稳状态。
      9. 向室的反应容器中加入1ml M9缓冲液,等待系统平衡
    4. 评估样品的氧气消耗率
      1. 在整个实验过程中启用搅拌。
      2. 使用M9缓冲液冲洗NGM板,并在15ml管中收集线虫
      3. 让动物在重力下沉降几分钟。使用成虫。去除大部分含有细菌,卵和幼虫的上清液(L1,L2,L3和L4)。重复此步骤两次。最后一次洗涤后,将体积减少至〜1.5 ml
      4. 从反应容器中取出M9缓冲液(见注3)
      5. 向室的反应容器中加入1ml线虫悬浮液
      6. 监测氧消耗5-10分钟。
      7. 准备下一个样品。洗去含有不同菌株或处理动物的其他平板。重复步骤D3-D5。
      8. 从反应容器中彻底回收动物,并将它们放在1.5ml管中(见注3)。
      9. 将管放在冰上。管子应保存在冰上,直到蛋白质测定
      10. 测量下一个样品。重复步骤D5-D9(见注4)。
      11. 保存从Oxygraph Plus软件记录的数据(图2B)。
      12. 从冰中收集样品,进行蠕虫裂解和蛋白质测定
      13. 将超声仪尖端插入样品,并使用十(10)脉冲,每次70%的功率对每个样品进行声波处理。立即将样品放在冰上(见注5)
      14. 在4℃下将样品在14,800×g下旋转10分钟。
      15. 将上清液(溶解的蛋白质)转移到新的1.5ml试管中,并使用标准蛋白试剂盒(如PierceBCA蛋白测定试剂盒)按照制造商的说明书测定蛋白质浓度(估计的蛋白质浓度范围:0.05 -0.9μg)。
      16. 打开并查看Microsoft Office 2011 Excel(图2B)中的数据(在步骤D11中获得)。
      17. 在Microsoft Office 2011 Excel软件中将负斜率(步骤D6中获得的氧消耗)与总蛋白量(nmol/min/ml/mg)的比率相除(图2C)。
      18. 将数据进行进一步和更高级的统计分析(图2C)
    5. 电极维护和存储
      1. 使用后清洁电极盘。
      2. 使用小棉球和维护套件中的抛光膏。
      3. 轻轻取出,去除通常在银电极上产生的氯化银和氧化盐(KCl)的棕色/黑色沉积(见注6)。重复抛光,直到电极表面上的所有棕色/黑色沉积物被清除
      4. 轻轻抛光电极圆顶的中心,避免刮伤。
      5. 用蒸馏水冲洗电极盘,清除抛光膏的所有痕迹(见注7)。
      6. 使用纸巾擦干电极盘。
      7. 将电极盘存放在密封容器中。

    数据分析

    1. 对于每个菌株或条件,使用至少100只动物,以获得更准确的结果。
    2. 对于每个实验,应当对每种菌株和条件检查相同数目的线虫。
    3. 每个测定应重复至少三(3)次。
    4. 使用学生的 t 测试,其显着性截止水平为 P 0.05用于两组之间的比较。
    5. 使用单因素(ANOVA)方差分析,并通过事后Bonferroni检验进行多重比较。

    笔记

    1. 不要拧得太紧,因为它可能会导致测量过程中的漂移信号过大拧紧大O型圈。
    2. 氧气电极盘对影响氧气水平的温度波动非常敏感。在整个实验期间保持实验温度稳定。在进行任何校准和/或测量之前,将电极盘平衡至测定温度
    3. 小心不要在缓冲液移除期间破坏电极盘的膜。
    4. 对每个应变或条件进行三次独立测量。
    5. 在超声处理期间,样品应保持冷以避免蛋白质降解。在超声处理期间不断地在冰上孵育样品
    6. 电极盘不应该在KCl就位的情况下干燥。 KCl残余物的结晶会氧化并破坏电极。将光盘维持在高标准是非常重要的
    7. 在维护过程中不要润湿光盘的电连接器。

    食谱

    1. 磷酸盐缓冲液(1M)
      1. 对于1L,在蒸馏水中溶解102.2g KH 2 PO 4和57.06g K 2 HPO 4,并且填充高达1 L.这是1M溶液,pH 6.0
      2. 高压灭菌并在室温下贮存
    2. 线虫生长培养基(NGM)琼脂平板
      1. 混合3g NaCl,2.5g Bacto TM蛋白胨,0.2g链霉素,17g琼脂并加入900ml蒸馏水。高压灭菌器
      2. 让温度降至55-60°C
      3. 加入1ml胆固醇储备溶液,1ml 1M CaCl 2,1ml 1M MgSO 4(无菌),1ml制霉菌素储备溶液,25ml无菌1M磷酸盐缓冲液,pH 6.0,和蒸馏无菌水至1L
      4. 每培养皿倒约8ml培养基,并固化
      5. 保持板在4°C,直到使用
    3. M9缓冲区
      1. 在1L蒸馏水中溶解3g KH 2 PO 4,6g Na 2 HPO 4,5g NaCl水。高压灭菌器
      2. 冷却并加入1ml 1M MgSO 4(无菌)
      3. 将M9缓冲液储存在4°C
    4. 氯化钾(KCl)缓冲液
      1. 将17.5g KCl溶于100ml蒸馏水中
      2. 将KCl缓冲液储存在4°C

    致谢

    这项工作由欧洲研究委员会(ERC),欧洲委员会7 框架计划和博多萨基金会博士后研究奖学金资助。该协议已经改编自Palikaras等人 。 (2015)Nature 521,525-528。

    参考文献

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    2. Chen,Z.,Wang,Z.,Guo,W.,Zhang,Z.,Zhao,F.,Zhao,Y.,Jia,D.,Ding,J.,Wang,H.,Yao,他,X.(2015)。 TRIM35与丙酮酸激酶同工型M2相互作用以抑制Warburg效应和致瘤性在肝细胞癌中。 癌基因 34(30):3946-3956。
    3. Dranka,BP,Benavides,GA,Diers,AR,Giordano,S.,Zelickson,BR,Reily,C.,Zou,L.,Chatham,JC,Hill,BG,Zhang,J.,Landar, -Usmar,VM(2011)。  评估生物能量功能通过代谢分析对氧化应激的反应。 Free Radic Biol Med 51(9):1621-1635。
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    7. Ryu,D.,Mouchiroud,L.,Andreux,PA,Katsyuba,E.,Moullan,N.,Nicolet-Dit-Felix,AA,Williams,EG,Jha,P.,Lo Sasso,G.,Huzard,D 。,Aebischer,P.,Sandi,C.,Rinsch,C.和Auwerx,J.(2016)。  尿囊素A诱导线粒体并延长秀丽隐杆线虫的寿命并增加啮齿类动物的肌肉功能。 Nat Med 22(8):879-888。
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    Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
    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.
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