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Intracellular Assessment of ATP Levels in Caenorhabditis elegans
秀丽隐杆线虫中细胞内ATP水平的评估   

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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. 1.5 ml tube
  2. Greiner Petri dishes (60 x 15 mm) (Greiner Bio One, catalog number: 628161 )
  3. Toothpick
  4. L4 larvae
  5. C. elegans strains (wild type [N2] and pink-1[tm1779])
  6. Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
  7. Liquid nitrogen
  8. Lyophilized ATP (provided with ATP bioluminescence assay kit CLS II) (Roche Diagnostics, catalog number: 11699695001 )
  9. Distilled water
  10. PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
  11. Glue
  12. 70% of EtOH
  13. ATP bioluminescence assay kit CLS II (Roche Diagnostics, catalog number: 11699695001 )
  14. Sodium chloride (NaCl) (EMD Millipore, catalog number: 1064041000 )
  15. BactoTM peptone (BD, catalog number: 211677 )
  16. Streptomycin (Sigma-Aldrich, catalog number: S-6501 )
  17. Agar (Sigma-Aldrich, catalog number: 05040 )
  18. Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
  19. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C-5080 )
  20. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M-7506 )
  21. Nystatin stock solution (Sigma-Aldrich, catalog number: N-3503 )
  22. Potassium dihydrogen phosphate (KH2PO4) (EMD Millipore, catalog number: 1048731000 )
  23. Na2HPO4 (EMD Millipore, catalog number: 1065860500 )
  24. K2HPO4
  25. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P-5405 )
  26. Phosphate buffer (1 M; sterile, see Recipes)
  27. Nematode growth medium (NGM) agar plates (see Recipes)
  28. M9 buffer (see Recipes)

Equipment

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

Software

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

Procedure

  1. Preparation of a synchronized nematode population
    1. Select L4 larvae of each strain by using a dissecting stereomicroscope. 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. 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 C. elegans 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 nematodes at the standard temperature of 20 °C.
    3. Four days later the plates contain mixed animals population.
    4. Synchronize nematodes by picking L4 larvae of each strain under a dissecting stereomicroscope and transfer them onto separate freshly E. coli (OP50) seeded plates (Figures 1A-1C).
    5. Add 20-25 L4 larvae per plate. For each experimental condition, use at least five plates.

  2. Preparation of nematodes samples
    1. Use an eyebrow/eyelash hair and collect 50-100 adult animals in 50 μl of M9 buffer in a 1.5 ml tube (see Note 1).
    2. Use the same number of animals of each strain during sample preparation.
    3. Freeze the samples in liquid nitrogen.
    4. Store the samples at -80 °C until further analysis.
    5. Immerse frozen worms in boiling water for 15 min.
    6. Place the samples on ice for 5 min.
    7. Spin down samples at 14,800 x g for 10 min at 4 °C.
    8. Transfer sample supernatants into new 1.5 ml tubes.
    9. Dilute sample supernatants tenfold by adding water before measurement.
    10. Keep the samples on ice or store them at -20 °C.

  3. Preparation of working solutions (ATP standards and luciferase reagent)
    1. ATP standards
      1. Dissolve lyophilized ATP by adding the appropriate volume of distilled water to get final concentration of 10 mg/ml or 16.5 mM (see Note 2).
      2. Store ATP standard solution at -20 °C. ATP standard solutions are stable for at least one week.
      3. Dilute ATP standards by serial dilutions in the range of 10 to 1 x 10-4 µM. Prepare serial dilutions of one ATP standard solution using distilled water to generate the ATP standard curve.
        Note: Diluted ATP standards solutions are stable for 8 h at 4 °C. Store them at -20 °C for longer periods of time.
    2. Luciferase reagent
      1. Dissolve lyophilized luciferase by adding 10 ml of distilled water.
      2. Incubate dissolved luciferase for 5 min at 4 °C.
      3. Gently swirl the bottle and mix for a homogeneous solution. Avoid shaking.
      4. Store luciferase reagent at -20 °C (see Note 3).

  4. Determination of ATP levels
    1. Add 100 μl of each sample or ATP standard in a 1.5 ml tube. Use a sample containing M9 buffer as blank (no ATP). For each sample or ATP standard, use at least 3 replicates.
    2. Add 100 μl luciferase reagent to the sample/standard (see Note 4).
    3. Incubate for 10 sec at room temperature.
    4. Measure and record ATP levels of the sample/standard by using TD-20/20 luminometer.
    5. Prepare and measure the next sample/standard. Repeat steps D1-D4.
    6. Transfer supernatants (solubilized proteins; obtained during step B8) of each sample 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 (Figure 2A).
    7. Open and review the recorded data (obtained during steps D4 and D6) in Microsoft Office 2011 Excel.
    8. Subtract the value of the ATP blank sample from the raw data.
    9. Prepare ATP standard curve by using the concentrations of ATP standards and their respective bioluminescence values.


      Figure 2. Intracellular measurement of ATP levels in C. elegans. A. Typical standard curve of bovine serum albumin (BSA) by using BCA protein determination assay. B. ATP standard curve using ATP bioluminescence assay kit CLS II. ATP dilutions were assayed with luciferase reagent in TD-20/20 luminometer. C. ATP levels are decreased in PINK-1 depleted nematodes (***P < 0.001; unpaired t-test). Error bars denote SEM.

    10. Calculate the ATP content of each sample by using a log-log plot of the ATP standard curve data (Figure 2B).
    11. Divided the ATP levels (obtained during step D10) with the total protein amount (nM/mg; obtained in step D6) in Microsoft Office 2011 Excel software.
    12. Subject the data to further and more advanced statistical analysis (Figure 2C).

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. Take a toothpick and glue an eyebrow/eyelash hair to the tip of it. Let it dry at room temperature. Then, use this tool to pick nematodes. Before using the eyebrow/eyelash hair always sterilize it by using 70% of EtOH.
  2. Each bottle of lyophilized ATP contains approximately 10 mg (> 98% purity; Mr: 605.2; ATP bioluminescence assay kit CLS II).
  3. Each freeze/thaw cycle reduces luciferase activity. Therefore, a. Avoid repeated freezing and thawing; b. Prepare aliquots of luciferase (1 ml); c. Set up a new ATP standard curve after each freeze/thaw cycle.
  4. Luciferase activity is influenced by extreme pH values. The optimal pH value for the luciferase reaction is between 7.6-8.0.

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 keep 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, 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. Store 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

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. de Wet, J. R., Wood, K. V., Helinski, D. R. and DeLuca, M. (1985). Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci U S A 82(23): 7870-7873.
  3. Drew, B. and Leeuwenburgh, C. (2003). Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction. Am J Physiol Regul Integr Comp Physiol 285(5): R1259-1267.
  4. Gomes, L. C., Di Benedetto, G. and Scorrano, L. (2011). During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nat Cell Biol 13(5): 589-598.
  5. Khakh, B. S. and Burnstock, G. (2009). The double life of ATP. Sci Am 301(6): 84-90, 92.
  6. Khan, H. A. (2003). Bioluminometric assay of ATP in mouse brain: Determinant factors for enhanced test sensitivity. J Biosci 28(4): 379-382.
  7. Lagido, C., Pettitt, J., Porter, A. J., Paton, G. I. and Glover, L.A. (2001). Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors. FEBS Lett 493(1): 36-39.
  8. McElroy, W. D. (1947). The energy source for bioluminescence in an isolated system. Proc Natl Acad Sci U S A 33(11): 342-345.
  9. Palikaras, K., Lionaki, E. and Tavernarakis, N. (2015). Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans. Nature 521(7553): 525-528.
  10. Vives-Bauza, C., Yang, L. and Manfredi, G. (2007). Assay of mitochondrial ATP synthesis in animal cells and tissues. Methods Cell Biol 80: 155-171.

简介

真核细胞严重依赖于由线粒体内的氧化磷酸化(OXPHOS)产生的三磷酸腺苷(ATP)。 ATP是主要能量货币分子,其促进细胞进行许多过程,包括生长,分化,运输和细胞死亡等(Khakh和Burnstock,2009)。因此,ATP水平可以作为细胞内稳态和存活的代谢指标(Artal-Sanz和Tavernarakis,2009; Gomes等人,2011; Palikaras等人, 2015)。在本议定书中,我们描述了使用生物发光方法在线虫秀丽隐杆线虫中测定细胞内ATP水平的方法。
关键词:老化,ATP,线粒体,能量稳态,荧光素酶,代谢,线粒体

[背景] 线粒体衍生的ATP在多种细胞和代谢中起着至关重要的作用过程。因此,已经开发了几种方法来测量这种重要代谢物的水平(Drew和Leeuwenburgh,2003; Khan,2003; Lagido等人,2001; Vives-Bauza等人, ,2007)。在1947年,McElroy提出使用萤火虫生物发光来确定细胞内ATP水平,当他发现ATP在光生产中的关键作用(McElroy,1947)。克隆和重组蛋白质技术的发展促进了萤火虫荧光素酶测定法的开发以测定ATP水平(de Wet等人,1985)。萤火虫荧光素酶,单体61 kD酶,催化荧光素的氧化,荧光素在560nm发光。当ATP是酶反应的限制因素时,光的强度与ATP的浓度成比例。因此,使用发光计允许检测光强度并随后确定ATP水平。在该协议中,我们描述了使用生物发光方法在ATP中ATP定量的方法。 elegans 。

关键字:衰老, ATP, 秀丽隐杆线虫, 能量稳态, 荧光素酶, 代谢, 线粒体

材料和试剂

  1. 1.5 ml管
  2. Greiner Petri dish(60×15mm)(Greiner Bio One,目录号:628161)
  3. 牙签
  4. L4幼虫
  5. C。 elegans 菌株(野生型[N2]和粉色-1 [tm1779] )
  6. 大肠杆菌 OP50菌株(获自Caenorhabditis Genetics Center)
  7. 液氮
  8. 冻干的ATP(与ATP生物发光测定试剂盒CLS II一起提供)(Roche Diagnostics,目录号:11699695001)
  9. 蒸馏水
  10. PierceBCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23225)
  11. 胶水
  12. 70%EtOH
  13. ATP生物发光测定试剂盒CLS II(Roche Diagnostics,目录号:11699695001)
  14. 氯化钠(NaCl)(EMD Millipore,目录号:1064041000)
  15. Bacto TM胨蛋白胨(BD,目录号:211677)
  16. 链霉素(Sigma-Aldrich,目录号:S-6501)
  17. 琼脂(Sigma-Aldrich,目录号:05040)
  18. 胆固醇储备溶液(SERVA电泳,目录号:17101.01)
  19. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C-5080)
  20. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M-7506)
  21. 制霉菌素储备液(Sigma-Aldrich,目录号:N-3503)
  22. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:1048731000)

  23. (EMD Millipore,目录号:1065860500)
  24. K 2 HPO 4
  25. 氯化钾(KCl)(Sigma-Aldrich,目录号:P-5405)
  26. 磷酸盐缓冲液(1M;无菌,参见配方)
  27. 线虫生长培养基(NGM)琼脂平板(见Recipes)
  28. M9缓冲区(请参阅配方)

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. TD-20/20发光计(Turner Designs,型号:2020-000)
  4. 台式离心机(Eppendorf,型号:5424)
  5. 冷冻机(西门子,-20℃; So-Low环境设备,型号:So-Low Ultra C85-22水平-80℃冷冻机)
  6. 加热板
  7. 火锅

软件

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

程序

  1. 同步线虫群体的制备
    1. 通过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. 秀丽隐杆线虫。 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.C的混合群体。通过解剖立体显微镜观察的线虫线虫。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. 在标准温度20℃下孵育线虫。
    3. 四天后,盘子含有混合动物种群
    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,并将它们转移到分离的新鲜E上。大肠杆菌(OP50)接种板(图1A-1C)
    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. 线虫样品的制备
    1. 使用眉毛/睫毛的头发,并收集50-100成年动物在50微升的M9缓冲液在1.5毫升管(见注1)。
    2. 在样品制备过程中使用相同数量的每种菌株的动物
    3. 在液氮中冷冻样品。
    4. 将样品储存在-80°C直到进一步分析
    5. 将冻虫浸泡在沸水中15分钟。
    6. 将样品在冰上放置5分钟
    7. 在4℃下将样品在14,800×g下旋转10分钟。
    8. 将样品上清液转移到新的1.5 ml试管中
    9. 在测量前加入水稀释样品上清液十倍
    10. 将样品保存在冰上或储存在-20°C
  3. 工作溶液(ATP标准品和萤光素酶试剂)的制备
    1. ATP标准品
      1. 通过添加适当体积的蒸馏水溶解冻干的ATP,使最终浓度为10 mg/ml或16.5 mM(参见注释2)。
      2. 将ATP标准溶液储存在-20°C。 ATP标准溶液至少稳定一周。
      3. 通过在10至1×10 4 -p μM范围内的系列稀释稀释ATP标准品。使用蒸馏水制备一个ATP标准溶液的系列稀释液,以产生ATP标准曲线 注意:稀释的ATP标准溶液在4°C下稳定8小时。将其在-20°C下存放更长时间。
    2. 萤光素酶试剂
      1. 加入10ml蒸馏水溶解冷冻干燥的荧光素酶
      2. 在4℃下孵育溶解的荧光素酶5分钟
      3. 轻轻旋转瓶子并混合均匀溶液。避免摇晃。
      4. 将萤光素酶试剂储存在-20℃(见注3)
  4. 确定ATP水平
    1. 添加100微升的每个样品或ATP标准在1.5毫升管。使用含有M9缓冲液的样品作为空白(无ATP)。对于每个样品或ATP标准,至少使用3次重复
    2. 向样品/标准品中加入100μl萤光素酶试剂(见注4)
    3. 在室温下孵育10秒。
    4. 使用TD-20/20光度计测量和记录样品/标准的ATP水平
    5. 准备并测量下一个样品/标准品。重复步骤D1-D4。
    6. 将每个样品的上清液(溶解的蛋白;在步骤B8期间获得)转移到新的1.5ml管中,并使用标准蛋白试剂盒如Pierce TM BCA蛋白测定试剂盒,根据制造商(图2A)
    7. 打开并查看Microsoft Office 2011 Excel中记录的数据(在步骤D4和D6中获得)。
    8. 从原始数据中减去ATP空白样品的值。
    9. 通过使用ATP标准品的浓度和它们各自的生物发光值制备ATP标准曲线

      图2.在C中的ATP水平的细胞内测量。 elegans 。 A.使用BCA蛋白测定法测定牛血清白蛋白(BSA)的典型标准曲线。 B.使用ATP生物发光测定试剂盒CLS II的ATP标准曲线。在TD-20/20发光计中用荧光素酶试剂测定ATP稀释液。 C.在PINK-1耗尽的线虫中ATP水平降低(***

      <0.001;未配对的测试)。误差线表示SEM

    10. 通过使用ATP标准曲线数据的对数图来计算每个样品的ATP含量(图2B)
    11. 在Microsoft Office 2011 Excel软件中将ATP水平(在步骤D10中获得)与总蛋白量(nM/mg;在步骤D6中获得)分开。
    12. 将数据进行进一步和更高级的统计分析(图2C)

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. 拿一根牙签,把眉毛/睫毛的头发粘到它的顶端。让它在室温下干燥。然后,使用此工具来选择线虫。在使用眉毛/睫毛之前,始终使用70%的EtOH对其进行消毒
  2. 每瓶冻干的ATP含有约10mg(纯度> 98%; Mr:605.2; ATP生物发光测定试剂盒CLS II)。
  3. 每个冻/融循环降低荧光素酶活性。因此,a。避免反复冻融; b。准备荧光素酶的等分试样(1ml); C。在每个冻/融循环后设置一个新的ATP标准曲线。
  4. 荧光素酶活性受极端pH值的影响。荧光素酶反应的最佳pH值在7.6-8.0之间

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. 高压灭菌并保持室温
  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 4,1ml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. 将板存储在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。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

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

  1. Artal-Sanz,M.和Tavernarakis,N。(2009)。  Prohibitin夫妇滞育信号在线虫体内老化期间线粒体代谢。 Nature 461(7265):793-797。
  2. de Wet,JR,Wood,KV,Helinski,DR和DeLuca,M。(1985)。  在大肠杆菌中克隆萤火虫荧光素酶cDNA和活性荧光素酶的表达。 ):7870-7873。
  3. Drew,B。和Leeuwenburgh,C。(2003)。  用于测量分离的线粒体中ATP产生的方法:具有年龄和热量限制的Fischer-344大鼠的脑和肝线粒体中的ATP产生。 Am J Physiol Regul Integr Comp Physiol 285(5) :R1259-1267。
  4. Gomes,LC,Di Benedetto,G.和Scorrano,L.(2011)。  Nat Cell Biol 13(5):589-598。
  5. Khakh,BS和Burnstock,G。(2009)。  ATP的双重寿命。 Sci Am 301(6):84-90,92。
  6. Khan,HA(2003)。  ATP的生物荧光测定法小鼠脑:用于增强测试灵敏度的决定因素。 J Biosci 28(4):379-382。
  7. Lagido,C.,Pettitt,J.,Porter,AJ,Paton,GI和Glover,LA(2001)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih .gov/pubmed/11278001"target ="_ blank">生物发光秀丽隐杆线虫作为多细胞真核生物传感器的开发和应用 FEBS Lett 493(1):36-39。 >
  8. McElroy,WD(1947)。  生物发光的能量源in a isolated system。 Proc Natl Acad Sci USA 33(11):342-345。
  9. Palikaras,K.,Lionaki,E.和Tavernarakis,N.(2015)。  在C中老化期间线粒体和线粒体生物发生的协调。 elegans 521(7553):525-528。
  10. Vives-Bauza,C.,Yang,L.和Manfredi,G.(2007)。  在动物细胞和组织中测定线粒体ATP合成。 方法细胞生物学 80:155-171。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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.
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