搜索

ATP and Lactate Quantification
ATP 和乳酸盐含量的定量测定

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Cells use glucose to generate energy by two different metabolic processes: lactic fermentation and aerobic respiration. In the first common series of reactions, glucose is converted into pyruvate. In anaerobic conditions, pyruvate is transformed into lactate, this process yields to 2 ATP molecules per glucose molecule. In the presence of oxygen, pyruvate is imported into mitochondria where it is used in the Krebs (or TCA) cycle and oxydative phosphorylation. The global process of oxydative phosphorylation yields to 32 ATP per glucose molecule. For reasons not fully understood, in some pathological cases like cancer, cells use anaerobic glycolysis even in the presence of oxygen, in which case the process is called aerobic glycolysis (or Warburg effect). This results in an increased uptake of glucose and lactate production. Measure of intracellular ATP content and lactate concentrations can provide a readout of aerobic glycolyis.

Materials and Reagents

  1. For intracellular ATP content evaluation
    1. Murine Embrionic Fibroblasts (MEF)
    2. Dulbecco’s Modified Eagle’s Medium High glucose with L-glutamine (DMEM) (Gibco®, catalog number: 11965 )
    3. Foetal Calf Serum (FCS) (Gibco®, catalog number: 16010 )
    4. Penicillin/Streptamycin (Gibco®, catalog number: 15070 )
    5. Phosphate Buffered Saline (PBS) (EuroClone, catalog number: E0B4004L )
    6. Protein Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
    7. Bovine Serum Albumine (BSA) (Sigma-Aldrich, catalog number: A4503 )
    8. NaCl
    9. Na4HPO4/NaH2PO4
    10. Glycerol
    11. Triton X-100
    12. Complete protease inhibitors (Roche Diagnostics, catlalog number: 11697498001 )
    13. Phosphatase inhibitors (1 mM final concentration of glycerophosphate, sodium orthovanadate and sodium fluoride)
    14. Complete protease inhibitors (Roche Diagnostics)
    15. ATP détermination kit (Life Technologies, catalog number: A22066 )
    16. Lysis buffer (see Recipes)
    17. Standard Reaction Solution (see Recipes)

  2. For Lactate quantification
    1. MEF
    2. EnzyChromTM L-lactate Assay Kit (BioAssay Systems, catalog number: ECLC-100 )
    3. Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Gibco®, catalog number: 11965)
    4. Foetal Calf Serum (FCS) (Gibco®, catalog number: 16010 )
    5. Penicillin/Streptamycin (Gibco®, catalog number: 15070 )
    6. 0.4% Trypan Blue stain (Life Technologies, catalog number: T10282 )

Equipment

  1. For intracellular ATP content evaluation
    1. 6 wells plates (Corning, Costar®, catalog number: CLS 3506 )
    2. Cell Scraper (Nunc®, catalog number: 179707 )
    3. Luminometer Gloomax 20/20
    4. Spectrophotometer
    5. 37 °C 5% CO2 Cell culture incubator
    6. Centrifuge spinning speed: 15682,186 G-force (13,000 rpm, 8,3 cm radius)

  2. For Lactate quantification
    1. 37 °C 5% CO2 Cell culture incubator
    2. 6 wells plates (Corning, Costar®, catalog number: CLS 3506)
    3. 96 wells plates (Corning, Costar®, catalog number: CLS 3596 )
    4. Countess automated cell counter with countess Cell Counting Chamber Slides (Life Technologies)
    5. Plate reader at 565 nm

Procedure

  1. For intracellular ATP content evaluation
    1. MEF are plated at 60-70% cell confluency.
    2. Cells are washed twice with PBS
    3. Cells are lysed with a cell scraper in 35 μl of lysis buffer containing phosphatase inhibitors (1 mM final concentration of glycerophosphate, sodium orthovanadate and sodium fluoride) for 30 min at 4 °C.
    4. Centrifuge at 13,000 rpm for 15 min at 4 °C.
    5. Supernatant is isolated.
    6. The sample is diluted at 1:1,000 (1 μl of sample is added to 1,000 μl of Biorad solution) in a Biorad solution (200 μl of Biorad and 800 μl of water).
    7. Optical Density (OD) is read at the spectrophotometer at 595 nm.
    8. The concentration of the sample is determined using a standard curve from BSA.
    9. Samples are diluted at a final concentration of 25 ng/ml.
    10. 250 ng of total proteins in parallel of the ATP standard curve (0 nM, 1 nM, 10 nM, 100 nM, 1,000 nM of ATP) in 10 μl is added to 90 μl the Standard Reaction Solution.
    11. Luciferase activity is measured with a luminometer.
    12.  ATP is quantified for each sample using the ATP standard curve.

  2. Lactate quantification
    1. Cells are plated in 6 wells plates with 3 ml of medium (10% DMEM-FCS-1% Penicillin/Streptamycin).
    2. The medium is changed when the cells reach 100% cell confluence.
    3. The medium is collected after 24 h.
    4. Cells are trypsinized and alive cells are counted with Trypan blue 1:1.
    5. The medium collected is centrifuged at 13,000 rpm for 10 min
    6. The supernatant (clean medium) is diluted 1:10 in DMEM.
    7. 20 μl of each sample and of the standard curve (0, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 and 1 mM of Lactate in DMEM) is put in wells of a 96 wells plate.
    8. 80 μl of assay buffer from EnzyChrom L-Lactate Assay Kit is added in each well.
    9. Mix well (Pipetting up and down).
    10. D0 is absorbed 565 nm at the beginning of the incubation ( t0) and after 20 min (t20).
    11. The difference of absorbtion (delta OD) is determined for the samples and the standard curve.
    12. The concentration of lactate for each sample is obtained from the standard curve.
    13.  The quantity of lactate is normalized by the final number of cells.

Recipes

  1. Lysis buffer
    150 mM NaCl
    20 mM Na4HPO4/NaH2PO4
    10% glycerol
    1% Triton X-100 (pH 7.2)
    Complete protease inhibitors
  2. Standard Reaction Solution
    1x Reaction Buffer
    0.1 mM DTT (dithiothreitol)
    0.5 ml 0.5 mM of D-luciferine
    2.5 μg/ml firefly luciferase and incubated at room temperature for 15 min (in the dark)

Acknowledgments

We thank all the co-authors of the article: Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H.,  Mari, S., Qian, F., Pei, Y. and Musco, G. and the other members of the lab Boletta.

References

  1. Rowe, I., Chiaravalli, M., Mannella, V., Ulisse, V., Quilici, G., Pema, M., Song, X. W., Xu, H., Mari, S., Qian, F., Pei, Y., Musco, G. and Boletta, A. (2013). Defective glucose metabolism in polycystic kidney disease identifies a new therapeutic strategy. Nat Med 19(4): 488-493.

简介

细胞使用葡萄糖通过两种不同的代谢过程产生能量:乳酸发酵和有氧呼吸。 在第一个常见的反应系列中,葡萄糖被转化为丙酮酸。 在厌氧条件下,丙酮酸转化为乳酸盐,该过程每个葡萄糖分子产生2个ATP分子。 在氧的存在下,丙酮酸进入线粒体,其用于Krebs(或TCA)循环和氧化磷酸化。 氧化磷酸化的全局过程产生每个葡萄糖分子32个ATP。 由于未完全理解的原因,在一些病理病例如癌症中,细胞即使在氧存在下也使用无氧糖酵解,在这种情况下,该过程称为有氧糖酵解(或沃伯格效应)。 这导致葡萄糖和乳酸盐产生的摄取增加。 细胞内ATP含量和乳酸盐浓度的测量可以提供有氧糖酵解的读出。

材料和试剂

  1. 用于细胞内ATP含量评价
    1. 鼠胚胎成纤维细胞(MEF)
    2. 用L-谷氨酰胺(DMEM)(Gibco ,目录号:11965)的Dulbecco改良Eagle培养基高葡萄糖
    3. 胎牛血清(FCS)(Gibco ,目录号:16010)
    4. 青霉素/链霉素(Gibco ,目录号:15070)
    5. 磷酸盐缓冲盐水(PBS)(EuroClone,目录号:E0B4004L)
    6. 蛋白染料试剂浓缩物(Bio-Rad Laboratories,目录号:500-0006)
    7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A4503)
    8. NaCl
    9. Na HPO 4/NaH <2> PO 4
    10. 甘油
    11. Triton X-100
    12. 完全蛋白酶抑制剂(Roche Diagnostics,catlalog number:11697498001)
    13. 磷酸酶抑制剂(1mM终浓度的甘油磷酸盐,原钒酸钠和氟化钠)
    14. 完全蛋白酶抑制剂(Roche Diagnostics)
    15. ATP终止试剂盒(Life Technologies,目录号:A22066)
    16. 裂解缓冲液(见配方)
    17. 标准反应溶液(参见配方)

  2. 乳酸盐定量
    1. MEF
    2. EnzyChrom TM L-乳酸盐测定试剂盒(BioAssay Systems,目录号:ECLC-100)
    3. Dulbecco改良的Eagle培养基具有稳定的L-谷氨酰胺(DMEM)的高葡萄糖(Gibco ,目录号:11965)
    4. 胎牛血清(FCS)(Gibco ,目录号:16010)
    5. 青霉素/链霉素(Gibco ,目录号:15070)
    6. 0.4%台盼蓝染料(Life Technologies,目录号:T10282)

设备

  1. 用于细胞内ATP含量评价
    1. 6孔板(Corning,Costar ,目录号:CLS 3506)
    2. 细胞刮刀(Nunc ,目录号:179707)
    3. 光度计Gloomax 20/20
    4. 分光光度计
    5. 37℃5%CO 2细胞培养箱
    6. 离心机旋转速度:15682,186G力(13,000rpm,8,3cm半径)

  2. 乳酸盐定量
    1. 37℃5%CO 2细胞培养箱
    2. 6孔板(Corning,Costar ,目录号:CLS 3506)
    3. 96孔板(Corning,Costar ,目录号:CLS 3596)
    4. 伯爵夫人自动细胞计数器与伯爵夫人细胞计数室幻灯片(生命技术)
    5. 读板仪在565 nm

程序

  1. 用于细胞内ATP含量评价
    1. MEF以60-70%细胞汇合铺板
    2. 用PBS洗涤细胞两次
    3. 将细胞用细胞刮刀在4℃下在含有磷酸酶抑制剂(1mM终浓度的甘油磷酸盐,原钒酸钠和氟化钠)的35μl裂解缓冲液中裂解30分钟。
    4. 在4℃下以13,000rpm离心15分钟
    5. 上清液是分离的。
    6. 在Biorad溶液(200μlBiorad和800μl水)中以1:1000稀释样品(1μl样品加入到1000μlBiorad溶液中)。
    7. 在分光光度计上在595nm读取光密度(OD)
    8. 使用来自BSA的标准曲线确定样品的浓度
    9. 将样品稀释至终浓度为25ng/ml
    10. 向90μl标准反应溶液中加入250ng总蛋白质(与ATP标准曲线(0nM,1nM,10nM,100nM,1,000nM ATP)平行)10μl。
    11. 用光度计测量荧光素酶活性
    12.  使用ATP标准曲线对每个样品的ATP进行定量。

  2. 乳酸定量
    1. 将细胞置于具有3ml培养基(10%DMEM-FCS-1%青霉素/链霉素)的6孔板中。
    2. 当细胞达到100%细胞汇合时,培养基更换
    3. 24小时后收集培养基。
    4. 将细胞胰蛋白酶化,并用台盼蓝1:1计数活细胞
    5. 收集的培养基在13,000rpm离心10分钟
    6. 将上清液(清洁培养基)在DMEM中以1:10稀释。
    7. 将20μl每种样品和标准曲线(0,0.1,0.2,0.3,0.4,0.6,0.8和1mM乳酸盐的DMEM溶液)置于96孔板的孔中。
    8. 将80μl来自EnzyChrom L-Lactate Assay Kit的测定缓冲液加入每个孔中。
    9. 混匀(向上和向下吸取)。
    10. 在孵育开始(t0)和20分钟(t20)后D0被吸收565nm
    11. 测定样品和标准曲线的吸光度差(ΔOD)
    12. 每个样品的乳酸盐浓度从标准曲线获得
    13.  乳酸盐的量通过细胞的最终数量归一化。

食谱

  1. 裂解缓冲液
    150mM NaCl 20mM Na 4 HPO 4 sub/NaH 2 PO 4 sub/4 NH 4 10%甘油 1%Triton X-100(pH7.2) 完全蛋白酶抑制剂
  2. 标准反应溶液
    1×反应缓冲液
    0.1mM DTT(二硫苏糖醇) 0.5ml 0.5mM D-荧光素
    2.5μg/ml萤火虫荧光素酶并在室温下孵育15分钟(在黑暗中)

致谢

我们感谢文章的所有共同作者:Chiaravalli,M.,Mannella,V.,Ulisse,V.,Quilici,G.,Pema,M.,Song,X.W.,Xu,H。 Mari,S.,Qian,F.,Pei,Y。和Musco,G。和实验室Boletta的其他成员。

参考文献

  1. Rowe,I.,Chiaravalli,M.,Mannella,V.,Ulisse,V.,Quilici,G.,Pema,M.,Song,XW,Xu,H.,Mari,S.,Qian,F.,Pei ,Y.,Musco,G。和Boletta,A。(2013)。 多囊肾病中的葡萄糖代谢不足确定了一种新的治疗策略。 Med 19(4):488-493。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Rowe, I., Chiaravalli, M. and Boletta, A. (2013). ATP and Lactate Quantification. Bio-protocol 3(23): e986. DOI: 10.21769/BioProtoc.986.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。