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Oct 2018
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Measurement of Respiration Rate in Live Caenorhabditis elegans
秀丽隐杆线虫活体呼吸速率的测定   

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Abstract

Mitochondrial function and dysfunction are at the core of aging and involved in many age-dependent diseases. Rate of oxygen consumption is a measure of mitochondrial function and energy production rate. The nematode Caenorhabditis elegans (C. elegans) offers an opportunity to study “living” mitochondria without the need for mitochondrial extraction, purification and associated artifacts. Oxygen consumption rate (OCR) is traditionally measured using single-chamber Clark electrodes with or without the addition of metabolic modulators. More recently, multi-well oxygen electrodes with automated injection system have been developed to enable rapid measurement of OCR under different conditions. Here, we describe a detailed protocol that we have adapted from existing protocols to measure coupled and uncoupled mitochondrial respiration (with and without metabolic modulators) in live respiring nematodes using a Seahorse XFe96 extracellular flux analyzer. We present details on our protocol, including preparation of nematode culture, use of metabolic modulators, execution of Seahorse XF assay as well as post-experimental data analysis. As a reference, we provide results of a series of experiments in which the metabolic activity of N2 wild-type nematodes was compared to N2 nematode treated with paraquat, a compound that generates reactive oxygen species (ROS), thus causing oxidative damage and mitochondrial dysfunction. These data illustrate the kind of insights that can be obtained even using a low number of nematodes (10 animals only per well).

Keywords: C. elegans (秀丽隐杆线虫), Nematodes (线虫), Mitochondrial respiration (线粒体呼吸), Mitochondrial metabolism (线粒体代谢), Seahorse XF analyzer (海马生命能量测定仪), Oxygen consumption rate (耗氧率), Oxidative phosphorylation (氧化磷酸化), Metabolic modulators (代谢调节剂)

Background

One key function of mitochondria is to produce adenosine triphosphate (ATP) via oxidative phosphorylation. Electrons are transferred along mitochondrial respiratory chain complexes with oxygen being the terminal electron acceptor (Voet et al., 2002). OCR is often used as proxy for energy production rate (Braeckman et al., 2002; Gruber et al., 2011; Fong et al., 2017). The development of multi-well oxygen electrodes with an automated injection system permits the study of up to 96 samples with automated addition of metabolic modulators during a single run. Due to the small volume of the chambers, OCR can also be established in live respiring C. elegans with only a small number of nematodes (10 animals per well), e.g., using the Seahorse XFe96 analyzer. Automated addition of mitochondrial uncoupler can be used to short-circuit mitochondrial membrane potential and accelerate transport of protons across the membrane thus permitting the observation of maximal respiratory capacity (Koopman et al., 2016; Fong et al., 2017; Huang and Lin, 2018). However, unlike in isolated cells, ATP synthase inhibitor does not have effect in C. elegans, at least in our case. Therefore, there is no measurement of ATP-linked respiration and proton leak, although others have suggested it may work in C. elegans but we cannot make this to work consistently (Luz et al., 2015). Temperature is another technical limitation in this assay as there is no temperature control in the Seahorse XF analyzer; the experiment has to be dependent on the environmental temperature where the XF analyzer is placed. Below, we will discuss details of the protocol used in our laboratory and also present our post-experimental data analysis and normalization workflow.

Materials and Reagents

  1. Pipette tips
  2. XFe96 sensor cartridges and tissue culture microplates (Agilent Technologies, Seahorse Bioscience, catalog number: 101085-004)
  3. 50 ml centrifuge tube (Greiner Bio-One, catalog number: 210261)
  4. Parafilm (Bemis, catalog number: HS234526C)
  5. C. elegans N2 Bristol (Caenorhabditis Genetics Center)
  6. OP50-1 E. coli bacteria (Caenorhabditis Genetics Center)
  7. Paraquat (Sigma-Aldrich, catalog number: 36541)
  8. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma-Aldrich, catalog number: C2920)
  9. Sodium azide (Sigma-Aldrich, catalog number: C2002)
  10. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650) 
  11. XF calibrant (Agilent Technologies, Seahorse Bioscience, catalog number: 100840-000)
  12. XFe96 Extracellular Flux Assay Kit (Agilent Technologies, Seahorse Bioscience, catalog number: 101085-004)
  13. NaCl (Sigma-Aldrich, catalog number: S5886)
  14. Bacto agar (BD Biosciences, catalog number: 214010)
  15. Bacteriological peptone (Sigma-Aldrich, catalog number: 91249)
  16. MgSO4 (Sigma-Aldrich, catalog number: M2643)
  17. CaCl2 (Sigma-Aldrich, catalog number: C5670)
  18. Cholesterol (Sigma-Aldrich, catalog number: C8667)
  19. Streptomycin (Sigma-Aldrich, catalog number: S9137)
  20. K2HPO4 (Sigma-Aldrich, catalog number: P3786)
  21. KH2PO4 (Sigma-Aldrich, catalog number: P5655)
  22. Na2HPO4 (Sigma-Aldrich, catalog number: S7907)
  23. NaOH (Sigma-Aldrich, catalog number: S8045)
  24. Nematode growth medium (NGM) agar (see Recipes)
  25. M9 buffer (see Recipes)
  26. Hypochlorite solution for worm synchronization (see Recipes)
  27. 1 M KPO4 (see Recipes)

Equipment

  1. Extracellular Flux Analyzer (Agilent Technologies, Seahorse Bioscience, model: XFe96)
  2. Non-CO2 incubator (Aqualytic, model: TC445S)

Software

  1. Seahorse Wave controller software (Agilent Technologies, Seahorse Bioscience) 
  2. Prism 8 (GraphPad Software, www.graphpad.com)

Procedure

  1. Synchronization of nematode population (Day 0) 
    1. Synchronize a plate of young adult nematodes with eggs (about 500 animals) using standard hypochlorite approach (Stiernagle, 2006).

  2. Growth of nematode population (Days 1 to 3)
    1. Maintain synchronized cohort consisting about 500 larvae in a 94 mm NGM agar plate in a 20 °C incubator until they reached adulthood (the synchronized wild-type L1 animals will reach young adult stage in about 50 h at 20 °C). 
    2. The day before transferring the young adult nematodes to the paraquat treatment plate, prepare the 10 mM paraquat treatment plate in advance by adding paraquat to the sterile NGM agar at 55 °C, and pour the NGM agar into a 94 mm plate and let the agar solidify. Seed the NGM agar plate with OP50-1 mixed with paraquat, dry the bacteria and keep the plate at 4 °C. 

  3. Paraquat treatment (Day 3)
    1. Transfer half of the young adults (about 250 nematodes) to paraquat treatment plate.

  4. Pick worms for assay (Day 4) 
    1. Pick the adult worms from control and treatment plates for oxygen measurement.

  5. Mitochondrial metabolic flux measurement
    Hydrate an XFe96 sensor cartridge (Day 3):
    1. Aliquot 20 ml of Seahorse XF Calibrant into a 50 ml centrifuge tube. 
    2. Place the centrifuge tube containing 20 ml of Seahorse XF calibrant in a non-CO2 37 °C incubator. 
    3. To hydrate the sensor cartridge of the XFe96 Extracellular Flux Assay Kit (Refer to Figure 1A)
      1. Remove the sensor cartridge from the utility plate and place the sensor cartridge upside down to make sure that the sensors do not contact with the surface of the lab bench. 
      2. Add 200 μl of sterile water into each well of the utility plate.
      3. Lower the sensor cartridge onto the utility plate to submerge the sensors in water. 
      4. Place the assembled sensor cartridge and utility plate in non-CO2 37 °C incubator overnight. If sensor cartridge is being hydrated more than 24 h, wrap parafilm around it to prevent evaporation. Hydrated sensor cartridge can be kept up to 72 h only. 
    4. Turn on the XFe96 controller (computer), XFe96 analyzer and start the wave controller software. Make sure the heater for the instrument is off and let the machine stabilize at room temperature or lower temperature overnight.


      Figure 1. Seahorse XFe96 sensor cartridge. (A) Top green plate is the sensor cartridge and bottom transparent plate is the utility plate. (B) To load the compound, place the compound loading guide plate on top of the sensor cartridge submerged in the utility plate filled with XF calibrant. Directly pipette the compound into the desired column of the compound loading guide plate to load the compounds into the sensor cartridge. Switch to the B/C loading guide plate when loading injection Port B or C.

    Day of Assay (Day 4)
    1. Hydrate an XFe96 sensor cartridge (continue from Day 3)
      1. Remove the assembled sensor cartridge and the tube containing 20 ml of the aliquoted XF Calibrant from the incubator. 
      2. Discard the water from the utility plate.
      3. Add 200 μl of the overnight per-warmed XF Calibrant into each well of the utility plate. 
      4. Lower the sensor cartridge onto the utility plate to submerge the sensors in water. 
      5. Place the assembled sensor cartridge and utility plate back to the non-CO2 37 °C incubator for 45 min.
    2. Preparation of drugs and loading of drugs into sensor cartridge injection ports
      1. During the 45 min incubation time, prepare the carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and sodium azide to be loaded into the drug ports. The FCCP and sodium azide concentrations have been optimized as stated in Fong et al. (2017). 
      2. Prepare a 2.5 mM stock of FCCP in 100% DMSO.
        1. Prepare 500 μl of FCCP dilution in M9 buffer to 90 μM. 
        2. Refer to Table 1 for FCCP stock concentration and dilutions.

          Table 1. FCCP stock and final concentration


      3. Prepare 5 M stock of sodium azide in MilliQ-H2O.
        1. Prepare 500 μl of sodium azide dilution in MilliQ-H2O to 500 mM. 
        2. Refer to Table 2 for sodium azide stock concentration and dilutions.

          Table 2. Sodium azide stock and final concentration


      4. After 45 min, load the compounds into the injection ports by using the A/D and B/C compound loading guide plates to dispense the compound into the injection port of the hydrated cartridge to prevent cross-contamination of compounds. 
      5. To load 25 μl of 90 μM FCCP into injection port A, place the A/D compound loading guide plate on top of the hydrate sensor cartridge. Make sure the letter A on the compound loading guide plate is located in the top left-hand corner. Use your thumb and index finger to hold the loading guide plate to secure it during loading so that the pipette tips do not dislodge from the loading guide plate. Refer to Figures 1B and 2 for the compound loading guide plates and the layout of the drug injection ports, respectively.
      6. Similarly, use B/C compound loading guide plate to load 25 μl of 500 mM Sodium Azide into injection port B. Refer to Figures 1B and 2 for the compound loading guide plates and the layout of the drug injection ports, respectively.
      7. Load the hydrated cartridge with 25 μl of 90 μM FCCP into port A and 25 μl of 500 mM Sodium Azide into Port B. Do not load the drugs into port A or B for well A1, A12, H1 and H12. Refer to Figures 1B and 2 for the compound loading guide plates and the layout of the drug injection ports, respectively.


        Figure 2. Layout of the drug injection ports in the XF sensor cartridge

      Notes:
      1. Ports A and B must be filled with the same volume of drugs or buffer. Ports C and D that are not in use can be kept empty. 
      2. Avoid creating air bubbles in the injection ports. 
      3. Do not tap the cartridge after loading the compounds to the injection ports as this will cause compound leakage from the injection port.

      1. Load 25 μl of M9 buffer into Port A of well A1, A12, H1 and H12 and 25 μl of MilliQ H2O into Port B of well A1, A12, H1 and H12. Well A1, A12, H1 and H12 are blank wells used for background correction. 
      2. Place the hydrated cartridge onto the utility plate filled with XF calibrant. 
      3. Set up the protocol using the “Wave controller” software.
      4. Carefully load the hydrated cartridge filled with compounds to the XFe96 seahorse analyzer. 
      5. Start the protocol to calibrate the hydrated sensor cartridge (approximately 40-50 min of calibration time).
    3. Transferring of C. elegans 
      1. During the calibration time, pipette 200 μl of M9 buffer into each well of a fresh utility plate. 
      2. Transfer 10 worms using worm pick into each well. The experiment must be done with at least 6 replicates for each condition. 
      3. For each plate, include 6 wells with the same amount of OP50 (food for worms) only. This serves as the control to measure baseline OCR derived from bacteria inside the worm gut.
      4. Refer to Figure 3 for plate layout below for the design of our experiment using N2 and N2 treated with 10 mM Paraquat.


        Figure 3. Plate layout for the design of our experiment using N2 and N2 treated with 10 mM Paraquat

    4. Running the seahorse XF assay
      1. Once the sensor cartridge calibration is completed, remove the calibration plate and replace with the utility plate containing worms in 200 μl of M9 buffer. 
      2. Remove the lid of the utility plate and place the plate into the XFe96 analyzer. 
      3. Start the equilibration. 
      4. After completing the equilibration, the assay will automatically begin to acquire baseline measurement according to the protocol that has been set earlier. 
      5. Use the protocol below to measure the mitochondrial respiration in C. elegans.
        Basal Respiration measurement:
        1. Loop 6 times
        2. Mix for 3 min 
        3. Time delay for 2 min 
        4. Measure for 3 min
        5. Loop end
        Inject Port A:
        1. Loop 6 times
        2. Mix for 3 min
        3. Time delay for 2 min
        4. Measure for 3 min
        5. Loop end
        Inject Port B:
        1. Loop 6 times
        2. Mix for 3 min 
        3. Time delay for 2 min 
        4. Measure for 3 min
        5. Loop end
      6. At the end of the run, save the data in the default XF cell energy phenotype assay result file (*.asyr) which can only be viewed using the XF wave software. Export the data to excel file (*.xlsx) and GraphPad (.pzfx) file for further analysis.

Data analysis

  1. Results were initially viewed in the type of kinetic graph from the XFe Analyser, Figure 4.


    Figure 4. Kinetic OCR graph. This experiment investigated mitochondrial metabolism in wild type N2, N2 treated with 10 mM Paraquat and oxygen consumption of bacteria used to culture the nematodes.

  2. Use the area under the curve of oxygen consumption rate (OCR), before and after the addition of compounds (FCCP followed by sodium azide) to determine basal respiration, maximal respiration and spare respiratory capacities. 
  3. Determine the basal respiration by determining the area under the curve (total OCR) from measurements 1 to 6 (prior to FCCP injection) by subtracting the non-mitochondrial respiration, area under the curve (total OCR) from measurements 13 to 18 (after sodium azide injection). Refer to Figure 5.


    Figure 5. Typical example of OCR profiles from a Seahorse experiment performed with C. elegans, replotted from OCR of N2 in Figure 3. Basal respiration rate was measured initially followed by FCCP injection, which allows the determination of the maximal and spare respiration rate.

  4. Determine the maximal respiration by determining the area under the curve (total OCR) from measurements 7 to 12 (prior to sodium azide injection) with subtracting the non-mitochondrial respiration 13 to 18 (after sodium azide injection). Refer to Figure 5.
  5. Determine the spare respiration by determining the area under the curve (total OCR) from measurements 6 to 18 (prior to sodium azide injection) with subtracting the basal respiration at measurements 1 to 6. Refer to Figure 5. 
  6. Normalize the basal respiration, maximal respiration and spare respiratory capacities to the number of worms in each well (10 worms per well). 
  7. Normalize the actual basal respiration, maximal respiration and spare respiratory capacities to the bacterial respiration rate. 
  8. lot the acquired data in bar graph to compare the respiration rate between treated and non-treated nematodes (Figures 6A-6C). 


    Figure 6. Bar graph comparing basal (A), maximal (B) and spare respiration (C) of wild type N2 and N2 treated with 10 mM Paraquat after our normalization workflow and post-experimental analysis. Data were plotted as means ± SEM, analyzed using Student's t-test. In the figures, P values ≤ 0.01 are summarized as two asterisks, P values ≤ 0.0001 are summarized as four asterisks.

Notes

  1. OCR values can differ between different runs due to different experimental conditions: time of worms in the buffer, changes in the temperature of the seahorse analyzer, different sensor cartridges, therefore, the experiment must be performed on the same plate. 
  2. There is no temperature control in the Seahorse XF analyzer, the heater has to be turned off and the analyzer has to be placed in a room with lower temperature. The experiment is now dependent on the environmental temperature and typically the temperature will increase by 2 to 4 °C during the run. We have tested the increase in the temperature and found that it does not cause significant effect in the OCR of the nematodes.

Recipes

  1. Nematode growth medium (NGM) agar
    For 1 L medium:
    3.0 g NaCl
    17.0 g Bacto agar
    2.5 g Bacteriological peptone
    Autoclave to sterilize the agar, cool to 55 °C then add:
    1 ml 1 M MgSO4 (filter to sterilize)
    1 ml 1 M CaCl2 (filter to sterilize)
    1 ml 5 mg/ml cholesterol in absolute ethanol (filter to sterilize)
    1 ml 200 mg/ml streptomycin (filter to sterilize)
    25 ml 1 M KPO4 (Recipe 4)
  2. M9 buffer (1 L)
    3 g KH2PO4
    6 g Na2HPO4
    5 g NaCl
    Autoclave, then add:
    1 ml 1 M MgSO4 (filter to sterilize)
  3. Hypochlorite solution for worm synchronization
    Prepare household bleach (5% solution of sodium hypochlorite) and 5 N NaOH in the ratio of 2:1
  4. 1 M KPO4 (1 L)
    108.3 g KH2PO4
    35.6 g K2HPO4
    H2O to 1 L
    Filter to sterilize

Acknowledgments

We thank the Caenorhabditis Genetics Centre for the provision of worm strains. Financial assistance from the Grant IG17-LR005 and IG17-BS101 is also acknowledged.

Competing interests

The authors declare that there are no conflicts of interest or competing interests.

References

  1. Braeckman, B. P., Houthoofd, K., De Vreese, A. and Vanfleteren, J. R. (2002). Assaying metabolic activity in ageing Caenorhabditis elegans. Mech Ageing Dev 123(2-3): 105-119.
  2. Fong, S., Ng, L. F., Ng, L. T., Moore, P. K., Halliwell, B. and Gruber, J. (2017). Identification of a previously undetected metabolic defect in the Complex II Caenorhabditis elegans mev-1 mutant strain using respiratory control analysis. Biogerontology 18(2): 189-200.
  3. Gruber, J., Ng, L. F., Fong, S., Wong, Y. T., Koh, S. A., Chen, C. B., Shui, G., Cheong, W. F., Schaffer, S., Wenk, M. R. and Halliwell, B. (2011). Mitochondrial changes in ageing Caenorhabditis elegans--what do we learn from superoxide dismutase knockouts? PLoS One 6(5): e19444. 
  4. Huang, S. H. and Lin, Y. W. (2018). Bioenergetic health assessment of a single Caenorhabditis elegans from postembryonic development to aging stages via monitoring changes in the oxygen consumption rate within a microfluidic device. Sensors (Basel) 18(8).
  5. Koopman, M., Michels, H., Dancy, B. M., Kamble, R., Mouchiroud, L., Auwerx, J., Nollen, E. A. and Houtkooper, R. H. (2016). A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans. Nat Protoc 11(10): 1798-1816.
  6. 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.
  7. Stiernagle, T. (2006). Maintenance of C. elegans. In: Fay, D and Ambros, V (Eds.). WormBook: the online review of C. elegans biology. 
  8. Voet, D., Voet, J. G. and Pratt, C. W. (2002). Fundamentals of Biochemistry: Life at the Molecular Level, 5th Edition. Vol. 2. John Wiley.

简介

摘要:线粒体功能和功能障碍是衰老的核心,并且涉及许多年龄依赖性疾病。耗氧率是线粒体功能和能量产生率的量度。线虫 Caenorhabditis elegans ( C.elegans )提供了研究“活的”线粒体的机会,而无需线粒体提取,纯化和相关的人工制品。传统上使用单腔Clark电极测量氧消耗速率(OCR),添加或不添加代谢调节剂。最近,已开发出具有自动注射系统的多孔氧电极,以便能够在不同条件下快速测量OCR。在这里,我们描述了一个详细的协议,我们已经改编自现有协议,使用海马XFe96细胞外通量分析仪测量活呼吸线虫的耦合和非耦合线粒体呼吸(有和没有代谢调节器)。我们提供了有关我们协议的详细信息,包括线虫培养的准备,代谢调节剂的使用,Seahorse XF测定的执行以及实验后数据分析。作为参考,我们提供了一系列实验的结果,其中将N2野生型线虫的代谢活性与用百草枯处理的N2线虫进行比较,百草枯是一种产生活性氧(ROS)的化合物,因此引起氧化损伤和线粒体功能障碍。 。这些数据说明了即使使用少量线虫也能获得的洞察力(每口仅10只动物)。

背景:线粒体的一个关键功能是通过氧化磷酸化产生三磷酸腺苷(ATP)。电子沿线粒体呼吸链复合物转移,氧是末端电子受体(Voet et al。,2002)。 OCR通常用作能源生产率的代理(Braeckman et al。,2002; Gruber et al。,2011; Fong et al。 ,2017)。利用自动注射系统开发多孔氧电极,可以在一次运行中自动添加代谢调节剂,最多可研究96个样品。由于腔室体积小,OCR也可以在现场呼吸 C中建立。使用Seahorse XFe96分析仪,只有少量线虫(每孔10只动物),例如的线虫。线粒体解偶联剂的自动添加可用于使线粒体膜电位短路并加速质子跨膜的转运,从而允许观察最大呼吸能力(Koopman 等。,2016; Fong 等人,,2017; Huang和Lin,2018)。然而,与分离的细胞不同,ATP合成酶抑制剂在 C中没有作用。线虫,至少在我们的案例中。因此,没有测量ATP连接的呼吸和质子泄漏,尽管其他人已经建议它可以在 C中起作用。线虫但我们无法使其始终如一地运作(Luz et al。,2015)。温度是该测定中的另一个技术限制,因为Seahorse XF分析仪中没有温度控制;实验必须取决于放置XF分析仪的环境温度。下面,我们将讨论我们实验室使用的协议的详细信息,并介绍我们的实验后数据分析和标准化工作流程。

关键字:秀丽隐杆线虫, 线虫, 线粒体呼吸, 线粒体代谢, 海马生命能量测定仪, 耗氧率, 氧化磷酸化, 代谢调节剂

材料和试剂

  1. 移液器吸头
  2. XFe96传感器盒和组织培养微孔板(Agilent Technologies,Seahorse Bioscience,目录号:101085-004)
  3. 50毫升离心管(Greiner Bio-One,目录号:210261)
  4. Parafilm(Bemis,目录号:HS234526C)
  5. ℃。线虫 N2布里斯托尔(Caenorhabditis遗传中心)
  6. OP50-1 E.大肠杆菌细菌(Caenorhabditis遗传中心)
  7. 百草枯(Sigma-Aldrich,目录号:36541)
  8. 羰基氰4-(三氟甲氧基)苯腙(FCCP)(西格玛奥德里奇,目录号:C2920)
  9. 叠氮化钠(Sigma-Aldrich,目录号:C2002)
  10. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650) 
  11. XF校准物(Agilent Technologies,Seahorse Bioscience,目录号:100840-000)
  12. XFe96细胞外通量检测试剂盒(Agilent Technologies,Seahorse Bioscience,目录号:101085-004)
  13. NaCl(Sigma-Aldrich,目录号:S5886)
  14. Bacto琼脂(BD Biosciences,目录号:214010)
  15. 细菌蛋白胨(Sigma-Aldrich,目录号:91249)
  16. MgSO 4 (Sigma-Aldrich,目录号:M2643)
  17. CaCl 2 (Sigma-Aldrich,目录号:C5670)
  18. 胆固醇(Sigma-Aldrich,目录号:C8667)
  19. 链霉素(Sigma-Aldrich,目录号:S9137)
  20. K 2 HPO 4 (Sigma-Aldrich,目录号:P3786)
  21. KH 2 PO 4 (Sigma-Aldrich,目录号:P5655)
  22. Na 2 HPO 4 (Sigma-Aldrich,目录号:S7907)
  23. NaOH(Sigma-Aldrich,目录号:S8045)
  24. 线虫生长培养基(NGM)琼脂(见食谱)
  25. M9缓冲液(见食谱)
  26. 用于蠕虫同步的次氯酸盐溶液(见食谱)
  27. 1 M KPO 4 (见食谱)

设备

  1. 细胞外通量分析仪(Agilent Technologies,Seahorse Bioscience,型号:XFe96)
  2. 非CO 2 培养箱(Aqualytic,型号:TC445S)

软件

  1. Seahorse Wave控制器软件(Agilent Technologies,Seahorse Bioscience) 
  2. Prism 8(GraphPad软件, www.graphpad.com )

程序

  1. 线虫种群同步(第0天) 
    1. 使用标准次氯酸盐法(Stiernagle,2006)使一盘年轻成虫线虫与卵(约500只动物)同步。

  2. 线虫种群的增长(第1天至第3天)
    1. 在20°C培养箱中维持一个94 mm NGM琼脂平板上约500只幼虫的同步组群,直到它们到达成年期(同步的野生型L1动物将在20°C下约50小时达到幼年期)。 
    2. 将幼年线虫转移到百草枯处理板的前一天,通过在55℃下向无菌NGM琼脂中加入百草枯,预先制备10mM百草枯处理板,并将NGM琼脂倒入94mm板中,然后将琼脂放入琼脂中。固化。将 OP50-1 与百草枯混合,将NGM琼脂平板接种,干燥细菌并将平板保持在4°C。

  3. 百草枯治疗(第3天)
    1. 将一半的年轻人(约250只线虫)转移到百草枯处理板上。

  4. 选择蠕虫进行检测(第4天) 
    1. 从控制和处理板中挑选成虫用于氧气测量。

  5. 线粒体代谢通量测量
    水合XFe96传感器盒(第3天):
    1. 将20毫升Seahorse XF Calibrant分装到50毫升离心管中。 
    2. 将含有20 ml Seahorse XF校准物的离心管放入非CO 2 37°C培养箱中。 
    3. 为XFe96细胞外通量检测试剂盒的传感器盒水合(参见图1A)
      1. 从实用板上取下传感器盒,将传感器盒倒置,确保传感器不与实验台的表面接触。 
      2. 在实用板的每个孔中加入200μl无菌水。
      3. 将传感器盒放在实用板上,将传感器浸入水中。 
      4. 将装配好的传感器盒和实用板放在37℃的非CO 2 培养箱中过夜。如果传感器盒正在水合超过24小时,请在其周围包裹封口膜以防止蒸发。水合传感器盒只能保持72小时。 
    4. 打开XFe96控制器(计算机),XFe96分析仪并启动波形控制器软件。确保仪器的加热器已关闭,让机器在室温或较低温度下稳定过夜。


      图1.海马XFe96传感器盒。(A)顶部绿色板是传感器盒,底部透明板是实用板。 (B)要装入化合物,将化合物装载导板放在浸没在装有XF校准物的实用板中的传感器盒顶部。将化合物直接吸移到化合物装载导板的所需柱中,以将化合物装入传感器盒中。装入进样口B或C时,切换到B / C装载导板。

    分析日(第4天)
    1. 水合XFe96传感器盒(从第3天开始)
      1. 从培养箱中取出装好的传感器盒和含有20 ml等分XF Calibrant的试管。 
      2. 丢弃实用板上的水。
      3. 将200μl过夜的每次温热的XF Calibrant加入实用板的每个孔中。 
      4. 将传感器盒放在实用板上,将传感器浸入水中。 
      5. 将装配好的传感器盒和实用板放回非CO 2 37°C培养箱中45分钟。
    2. 制备药物并将药物装入传感器盒注射口
      1. 在45分钟的孵育时间内,制备羰基氰化物对 - (三氟甲氧基)苯腙(FCCP)和叠氮化钠,将其装入药物端口。如Fong 等(2017)所述,FCCP和叠氮化钠浓度已经过优化。 
      2. 在100%DMSO中制备2.5mM FCCP储备液。
        1. 在M9缓冲液中制备500μlFCCP稀释液至90μM。 
        2. 有关FCCP原料浓度和稀释度,请参阅表1。

          表1. FCCP库存和最终浓度


      3. 在MilliQ-H 2 O中制备5M叠氮化钠。
        1. 在MilliQ-H 2 O中制备500μl叠氮化钠稀释液至500 mM。 
        2. 关于叠氮化钠原料浓度和稀释度,请参阅表2。

          表2.叠氮化钠原料和最终浓度


      4. 45分钟后,使用A / D和B / C化合物装载导板将化合物装入进样口,将化合物分配到水合滤芯的进样口,以防止化合物交叉污染。 
      5. 要将25μl的90μMPFC加载到进样口A,将A / D化合物加载导板放在水合物传感器盒的顶部。确保复合装载导板上的字母A位于左上角。使用拇指和食指握住装载导板,以便在装载过程中将其固定,以使移液器吸头不会从装载导板上脱落。有关复合装载导板和药物注射口的布局,请分别参见图1B和图2。
      6. 类似地,使用B / C化合物装载导板将25μl500mM叠氮化钠装入注射口B.分别参见图1B和2,了解化合物装载导板和药物注射口的布局。
      7. 将含有25μl90μMFCCP的水合滤芯装入A口,将25μl500mM叠氮化钠装入B口。不要将药物装入A1,A12,H1和H12井的A口或B口。有关复合装载导板和药物注射口的布局,请分别参见图1B和图2。


        图2. XF传感器盒中药物注射端口的布局

      注意:
      1. 端口A和B必须填充相同数量的药物或缓冲液。未使用的端口C和D可以保持为空。 
      2. 避免在进样口产生气泡。 
      3. 在将化合物装入进样口后不要轻敲药筒,因为这会导致化合物从进样口泄漏。

      1. 将25μlM9缓冲液加载到孔A1,A12,H1和H12的端口A和25μlMilliQH 2 O中,进入孔A1,A12,H1和H12的端口B. A1,A12,H1和H12井是用于背景校正的空白孔。 
      2. 将含水墨盒放在装有XF校准液的实用板上。 
      3. 使用“Wave controller”软件设置协议。
      4. 小心地将装有化合物的水合墨盒装入XFe96海马分析仪。 
      5. 启动协议以校准水合传感器盒(校准时间约为40-50分钟)。
    3. 转移 C.线虫 
      1. 在校准期间,将200μlM9缓冲液吸移到新鲜实用板的每个孔中。 
      2. 使用蠕虫镐将10只蠕虫转移到每个孔中。对于每种情况,必须至少进行6次重复实验。 
      3. 对于每个平板,包括6个具有相同量的OP50(蠕虫食物)的孔。这用作测量来自蠕虫肠内细菌的基线OCR的对照。
      4. 关于使用用10mM百草枯处理的N2和N2进行实验设计,请参考图3中的板布局。


        图3.使用10 mM百草枯处理N2和N2进行实验设计的平板布局

    4. 运行海马XF测定
      1. 完成传感器盒校准后,取下校准板并更换含有200μlM9缓冲液中的蠕虫的实用板。 
      2. 取下实用板的盖子,将平板放入XFe96分析仪中。 
      3. 开始平衡。 
      4. 完成平衡后,测定将根据之前设定的方案自动开始获得基线测量。 
      5. 使用以下方案测量 C中的线粒体呼吸。线虫。
        基础呼吸测量
        1. 循环6次
        2. 混合3分钟 
        3. 延时2分钟 
        4. 测量3分钟
        5. 循环结束
        注入端口A:
        1. 循环6次
        2. 混合3分钟
        3. 延时2分钟
        4. 测量3分钟
        5. 循环结束
        注入端口B:
        1. 循环6次
        2. 混合3分钟 
        3. 延时2分钟 
        4. 测量3分钟
        5. 循环结束
      6. 在运行结束时,将数据保存在默认的XF细胞能量表型分析结果文件(* .asyr)中,该文件只能使用XF波形软件查看。将数据导出到excel文件(* .xlsx)和GraphPad(.pzfx)文件以进行进一步分析。

数据分析

  1. 结果最初是从XFe分析仪的动力学图表类型中查看的,图4.


    图4.动力学OCR图。该实验研究了用10mM百草枯处理的野生型N2,N2处理的线粒体代谢和用于培养线虫的细菌耗氧量。

  2. 在加入化合物(FCCP后加叠氮化钠)之前和之后,使用氧消耗率曲线下面积(OCR),以确定基础呼吸,最大呼吸和备用呼吸能力。 
  3. 通过从测量值13到18(钠后)减去非线粒体呼吸,曲线下面积(总OCR),确定测量1到6(FCCP注射前)的曲线下面积(总OCR)来确定基础呼吸叠氮化物注射)。参见图5.


    图5.使用 C执行的Seahorse实验的OCR配置文件的典型示例。线虫,从图3中N2的OCR重新绘制。最初测量基础呼吸率,然后进行FCCP注射,这样可以确定最大和备用呼吸率。

  4. 通过从测量7至12(叠氮化钠注射之前)确定曲线下面积(总OCR)并减去非线粒体呼吸13至18(叠氮化钠注射后)来确定最大呼吸。参见图5。
  5. 通过测量从测量值6到18(叠氮化钠注射之前)的曲线下面积(总OCR)确定备用呼吸,并在测量1到6处减去基础呼吸。参见图5. 
  6. 将基础呼吸,最大呼吸和备用呼吸能力标准化为每口井中的蠕虫数量(每孔10个蠕虫)。 
  7. 将实际基础呼吸,最大呼吸和备用呼吸能力标准化为细菌呼吸率。 
  8. 将条形图中获得的数据用于比较治疗线虫和未治疗线虫之间的呼吸率(图6A-6C)。 


    图6.比较我们的标准化工作流程和实验后分析后用10 mM百草枯处理的野生型N2和N2的基础(A),最大(B)和备用呼吸(C)的条形图。数据将其绘制为平均值±SEM,使用Student's t -test进行分析。在图中, P 值≤0.01总结为两个星号, P 值≤0.0001总结为四个星号。

笔记

  1. 由于不同的实验条件,不同运行的OCR值可能不同:缓冲液中的蠕虫时间,海马分析仪的温度变化,不同的传感器盒,因此,实验必须在同一板上进行。 
  2. Seahorse XF分析仪没有温度控制,必须关闭加热器,并且必须将分析仪放在温度较低的房间内。该实验现在取决于环境温度,并且通常在运行期间温度将增加2至4℃。我们测试了温度的升高,发现它不会对线虫的OCR产生显着影响。

食谱

  1. 线虫生长培养基(NGM)琼脂
    对于1升介质:
    3.0克NaCl
    17.0克Bacto琼脂
    2.5克细菌蛋白胨
    高压灭菌器对琼脂进行灭菌,冷却至55°C,然后加入:
    1毫升1M MgSO 4 (过滤灭菌)
    1毫升1M CaCl 2 (过滤灭菌)
    1毫升5毫克/毫升胆固醇在无水乙醇(过滤消毒)
    1毫升200毫克/毫升链霉素(过滤灭菌)
    25毫升1M KPO 4 (配方4)
  2. M9缓冲液(1升)
    3克KH 2 PO 4
    6g Na 2 HPO 4
    5克NaCl
    高压灭菌器,然后添加:
    1毫升1M MgSO 4 (过滤灭菌)
  3. 用于蠕虫同步的次氯酸盐溶液
    准备家用漂白剂(5%次氯酸钠溶液)和5 N NaOH,比例为2:1
  4. 1 M KPO 4 (1 L)
    108.3g KH 2 PO 4
    35.6g K 2 HPO 4
    H 2 O至1 L
    过滤消毒

致谢

我们感谢Caenorhabditis遗传中心提供蠕虫菌株。 Grant IG17-LR005和IG17-BS101的财政援助也得到了承认。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

  1. Braeckman,B.P.,Houthoofd,K.,De Vreese,A。和Vanfleteren,J.R。(2002)。 测定衰老中的新陈代谢活动 Caenorhabditis elegans 。 Mech Aging Dev 123(2-3):105-119。
  2. Fong,S.,Ng,L.F.,Ng,L.T.,Moore,P.K.,Halliwell,B.and Gruber,J。(2017)。 鉴定复合体II Caenorhabditis elegans mev-1中先前未检测到的代谢缺陷 Biogerontology 18(2):189-200。
  3. Gruber,J.,Ng,LF,Fong,S.,Wong,YT,Koh,SA,Chen,CB,Shui,G.,Cheong,WF,Schaffer,S.,Wenk,MR and Halliwell,B。(2011 )。 衰老中的线粒体变化 Caenorhabditis elegans - 我们从超氧化物中学到了什么? dismutase knockouts? PLoS One 6(5):e19444。 
  4. Huang,S.H。和Lin,Y.W。(2018)。 从胚后发育到衰老阶段的单一 Caenorhabditis elegans 的生物能量健康评估通过监测微流体装置内氧气消耗速率的变化。 传感器(巴塞尔) 18(8)。
  5. Koopman,M.,Michels,H.,Dancy,B.M.,Kamble,R.,Mouchiroud,L.,Auwerx,J.,Nollen,E.A。和Houtkooper,R.H。(2016)。 基于筛选的平台,用于评估秀丽隐杆线虫中的细胞呼吸 。 Nat Protoc 11(10):1798-1816。
  6. Luz,A.L.,Rooney,J.P.,Kubik,L.L。,Gonzalez,C.P.,Song,D。H.和Meyer,J.N。(2015)。 线粒体呼吸链的线粒体形态和基本参数在 Caenorhabditis elegans中被改变缺乏线粒体动力学和稳态过程的菌株。 PLoS One 10(6):e0130940。
  7. Stiernagle,T。(2006)。 C的维护。线虫。在:Fay,D和Ambros,V(编辑)。 WormBook: C的在线评论。线虫生物学。 
  8. Voet,D.,Voet,J。G.和Pratt,C.W。(2002)。 生物化学基础:生命在分子水平,第5版。 卷。 2. John Wiley。
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引用:Ng, L. F. and Gruber, J. (2019). Measurement of Respiration Rate in Live Caenorhabditis elegans. Bio-protocol 9(10): e3243. DOI: 10.21769/BioProtoc.3243.
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