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Determination of H2O2 Generation by pHPA Assay
采用pHPA实验测定H2O2生成   

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Abstract

The production of reactive oxygen species, including H2O2, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H2O2 release from cells (Panus et al., 1993; Murthy et al., 2010; Larson-Casey et al., 2016; Larson-Casey et al., 2014; He et al., 2011). This assay takes advantage of H2O2-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes p-hydroxyphenylacetic acid (pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5’ diacetate [(pHPA)2]). The H2O2-dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H2O2 release from cells. This assay can measure H2O2 release from whole cells, mitochondria, or the NADPH oxidase.

Background

H2O2 generation primarily results from dismutation of superoxide anion (O2-), which occurs at a rapid rate (105-106 M-1 s-1) non-enzymatically. Unlike O2-, H2O2 can traverse membranes easily, so it is able to oxidize multiple molecules. ROS can be toxic to cells by oxidizing proteins, lipids, and nucleic acids and are associated with many human diseases. This protocol allows for detection of H2O2 release from NADPH oxidase or mitochondria in various cell types.

Materials and Reagents

  1. NuncTM 96-well black bottom plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 137101 )
  2. Eppendorf tubes
  3. 15 and 50 ml conical tubes
  4. Glucose (Sigma-Aldrich, catalog number: G7528 )
  5. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080 )
  6. Sodium bicarbonate (NaHCO3) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S233-500 )
  7. 4-hydroxyphenylacetic acid (pHPA) (Sigma-Aldrich, catalog number: H50004 )
  8. HRP (Sigma-Aldrich, catalog number: P8125 )
  9. Ca2+/Mg2+/phenol red-free HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14175-095 )
  10. α-Ketoglutaric acid (Sigma-Aldrich, catalog number: K2000 )
  11. Hydrogen peroxide solution (H2O2) (30%, w/w) (Sigma-Aldrich, catalog number: H1009 )
  12. Tris
  13. EDTA
  14. Sucrose
  15. Protease inhibitor tablets (Sigma-Aldrich, catalog number: 11836170001 )
  16. Phosphatase inhibitors (EMD Millipore, catalog number: 524625 )
  17. Antimycin A (optional)
  18. MitoTempo (optional)
  19. pHPA buffer (see Recipes)
  20. 1 mM H2O2 working stock solution (see Recipes)
  21. Mitochondrial buffer (see Recipes)

Equipment

  1. Kontes pellet pestle motor
  2. Centrifuge
  3. Plate reader (Molecular Devices, model: M2 SpectraMax )

Procedure

  1. Prepare pHPA buffer (see Recipes)
  2. Prepare H2O2 standard curve (see Figure 1)
    Note: Prepared in pHPA buffer prior to the assay (The buffer cannot be stored for subsequent measurements, make fresh at time of use).
    1. 8-12 standards in duplicate or triplicate.
    2. Load standards into 96-well plate.
      Highest concentration of H2O2: 2 µM (2 µl of 1 mM H2O2 working stock solution/ml pHPA buffer)
      Serial dilution: 50%
      #wells/standard: 2 or 3
      µl/well: 200
       

      Figure 1. Schematic of 96-well plate with 12 standards (in duplicate) and samples (in triplicate)

  3. Prepare samples
    1. Sample isolation
      1. Samples can be whole cells, freshly isolated mitochondria or membrane fractions from various cell types (i.e., macrophages, fibroblasts, bronchoalveolar lavage cells, etc.).
      2. Mitochondrial isolation:
        1) Lyse 10 million macrophages in (100 µl) mitochondrial buffer (see Recipes). Lysis is performed using Kontes Pellet Pestle Motor for 30 sec to homogenize each sample.
        2) Centrifuge at 2,000 x g for 10 min at 4 °C, save supernatant at 4 °C.
        3) Pellet is lysed and centrifuged [repeat steps 1) and 2)].
        4) The two supernatants are pooled and centrifuged at 12,000 x g for 15 min at 4 °C.
        5) The pellet is resuspended in (50 µl) mitochondrial buffer without sucrose [same as in 1) but do not add sucrose].
    2. Each sample should be run in duplicate or triplicate.
    3. Load samples into 96-well plate:
      1. Use 5-20 µg protein or 50,000-100,000 cells per well
      2. Suspend sample in pHPA buffer, mix well
      3. Load 200 µl/well in duplicate
    4. Positive controls may be added (i.e., treat cells with 100 μM antimycin A).
    5. Negative controls may be added (i.e., treat cells with 10 μM MitoTempo).
  4. Run assay in plate reader
    1. Pre-warm plate reader to 37 °C.
    2. Insert plate.
    3. Run kinetic assay, typically for 4 h with a read every 5-10 min.
    4. Use an excitation wavelength of 320 nm and emission of 400 nm.

Data analysis

  1. Use initial readings of standards to generate a standard curve.
  2. Determine the H2O2 concentration in samples by applying a linear regression to the standard curve and extrapolate.
  3. Normalize samples to protein or cell number. Samples can be lysed in protein sample buffer to determine cellular protein levels, or alternatively when using live cells, the number of cells can be used to normalize data.
  4. Data can be expressed as a rate over a set time (Figure 2A) or by showing a kinetic curve (Figure 2B); also see Larson-Casey et al., 2016.


    Figure 2. Mitochondrial H2O2 generation in macrophages expressing empty or constitutively active Akt1 (Akt1CA) expression vectors

Notes

  1. This assay is typically performed on freshly isolated mitochondrial (see Recipes for protocol) or membrane factions.
  2. Prepare all working solutions on day of use and do not reuse.

Recipes

  1. pHPA buffer
    50 ml total volume made up in Ca2+/Mg2+/phenol red-free HBSS
    Make up buffer in 50 ml conical tube and protect from light

  2. 1 mM H2O2 working stock solution
    1.13 µl 30% (w/w) H2O2 stock solution/10 ml H2O
    Note: H2O2 MW = 84.01, 30% (w/w) (H2O2 stock solution) equivalent to 8.82 M.
  3. Mitochondrial buffer
    10 mM Tris, pH 7.8
    0.2 mM EDTA
    320 mM sucrose
    1 protease inhibitor tablet
    Phosphatase inhibitors diluted 1:100

Acknowledgments

This work was supported by 2R01ES015981 & VA merit review BX001135. This protocol was originally adapted from Panus et al. (1993).

References

  1. He, C., Murthy, S., McCormick, M. L., Spitz, D. R., Ryan, A. J. and Carter, A. B. (2011). Mitochondrial Cu,Zn-superoxide dismutase mediates pulmonary fibrosis by augmenting H2O2 generation. J Biol Chem 286(17): 15597-15607.
  2. Larson-Casey, J. L., Deshane, J. S., Ryan, A. J., Thannickal, V. J. and Carter, A. B. (2016). Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis. Immunity 44(3): 582-596.
  3. Larson-Casey, J. L., Murthy, S., Ryan, A. J. and Carter, A. B. (2014). Modulation of the mevalonate pathway by Akt regulates macrophage survival and development of pulmonary fibrosis. J Biol Chem 289(52): 36204-36219.
  4. Murthy, S., Ryan, A., He, C., Mallampalli, R. K. and Carter, A. B. (2010). Rac1-mediated mitochondrial H2O2 generation regulates MMP-9 gene expression in macrophages via inhibition of SP-1 and AP-1. J Biol Chem 285(32): 25062-25073.
  5. Panus, P. C., Radi, R., Chumley, P. H., Lillard, R. H. and Freeman, B. A. (1993). Detection of H2O2 release from vascular endothelial cells. Free Radic Biol Med 14(2): 217-223.

简介

包括H 2 O 2 O 2的活性氧物质的产生是可用于信号传导,细胞死亡或免疫应答的过程。为了量化细胞中的氧化应激,已经从先前描述的方法修改了荧光技术以测量来自细胞的H 2 O 2 O 2释放(Panus等人, 2011; Larson-Casey等人,1993; Murthy等人,2010; Larson-Casey等人,2016; Larson-Casey等人 2014; He 等人,2011)。该测定利用了辣根过氧化物酶(HRP)对配合物I的H 2 O 2 O 2 - 介导的氧化,这又将对羟基苯乙酸(pHPA)氧化成稳定的荧光pHPA二聚体(2,2'-二羟基 - 联苯-5,5'-二乙酸酯[(pHPA)2])。 H 2 O 2 O 2依赖性HRP介导的pHPA氧化是用于定量H 2 O 2 O 2的灵敏和特异性的测定,/sub>从细胞释放。该测定可以测量来自全细胞,线粒体或NADPH氧化酶的H 2 O 2亚型释放。

[背景] (O 2→O 2 - )的超导阴离子(O 2 2-)的歧化导致的强烈的H 2 O 2 O 2 - 非 - 酶促反应,其中所述反应混合物是非酶促的。与O 2 - 不同,H 2 O 2可以容易地穿过膜,因此其能够氧化多个分子。 ROS可以通过氧化蛋白质,脂质和核酸而对细胞有毒并且与许多人类疾病相关。该方案允许检测在各种细胞类型中从NADPH氧化酶或线粒体的H 2 O 2 O 2释放。

材料和试剂

  1. Nunc 96孔黑色底板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:137101)
  2. Eppendorf管
  3. 15和50ml锥形管
  4. 葡萄糖(Sigma-Aldrich,目录号:G7528)
  5. HEPES(1μM)(Thermo Fisher Scientific,Gibco TM ,目录号:15630-080)
  6. 碳酸氢钠(NaHCO 3)(Thermo Fisher Scientific,Fisher Scientific,目录号:S233-500)
  7. 4-羟基苯基乙酸(pHPA)(Sigma-Aldrich,目录号:H50004)
  8. HRP(Sigma-Aldrich,目录号:P8125)
  9. (Thermo Fisher Scientific,Gibco< sup>,目录号:14175-095)的H 2 SO 4/Ca 2+ /酚/Mg 2+ br />
  10. α-酮戊二酸(Sigma-Aldrich,目录号:K2000)
  11. 过氧化氢溶液(H 2 O 2 sub)(30%,w​​/w)(Sigma-Aldrich,目录号:H1009)
  12. Tris
  13. EDTA,
  14. 蔗糖
  15. 蛋白酶抑制剂片剂(Sigma-Aldrich,目录号:11836170001)
  16. 磷酸酶抑制剂(EMD Millipore,目录号:524625)
  17. 抗霉素A(可选)
  18. MitoTempo(可选)
  19. pHPA缓冲液(参见配方)
  20. 1mM H 2 O 2工作原液(参见配方)
  21. 线粒体缓冲液(见配方)

设备

  1. 控制颗粒杵电机
  2. 离心机
  3. 读板器(Molecular Devices,型号:M2 SpectraMax)

程序

  1. 准备pHPA缓冲液(参见配方)
  2. 准备H sub 2 O 2标准曲线(参见图1)
    注意:在测定之前在pHPA缓冲液中制备(缓冲液不能存储用于后续测量,在使用时更新)。
    1. 8-12标准,一式两份或一式三份
    2. 将标准品装入96孔板。
      最高浓度的H 2 O 2 O 2:2μM(2μl的1mM H 2 O 2 O 2工作油料溶液/ml pHPA缓冲液) 系列稀释:50%
      #wells/standard:2或3
      μl/孔:200μg/孔  

      图1.具有12个标准品(一式两份)和样品(一式三份)的96孔板示意图

  3. 准备样品
    1. 样本隔离
      1. 样品可以是全细胞,新鲜分离的线粒体或来自各种细胞类型(即巨噬细胞,成纤维细胞,支气管肺泡灌洗细胞等)的膜级分。
      2. 线粒体隔离:
        1)在(100μl)线粒体缓冲液中溶解1000万巨噬细胞(参见Recipes)。使用Kontes Pellet Pestle Motor进行30秒的裂解以均质化每个样品 2)在4℃下以2,000xg离心10分钟,在4℃下保存上清液。
        3)将沉淀裂解并离心[重复步骤1)和2)] 4)将两种上清液合并,并在4℃下以12,000xg离心15分钟。
        5)将沉淀物重悬于不含蔗糖(与1中相同)的(50μl)线粒体缓冲液中,但不添加蔗糖]。
    2. 每个样品应重复或重复运行。
    3. 将样品装入96孔板:
      1. 每孔使用5-20μg蛋白或50,000-100,000个细胞,
      2. 将样品悬浮在pHPA缓冲液中,混匀
      3. 加载200μl/孔,一式两份
    4. 可以加入阳性对照(,。)用100μM抗霉素A处理细胞。
    5. 可以加入阴性对照(,即。用10μMMitoTempo处理细胞)。
  4. 在读板器
    中运行测定
    1. 将预温读板机预温至37°C
    2. 插入板。
    3. 运行动力学测定,通常为4小时,每5-10分钟读一次
    4. 使用320nm的激发波长和400nm的发射。

数据分析

  1. 使用标准的初始读数生成标准曲线。
  2. 通过对标准曲线和外推进行线性回归来确定样品中的H 2 O 2 O 2浓度。
  3. 将样品标准化为蛋白质或细胞数。样品可以在蛋白质样品缓冲液中裂解以确定细胞蛋白质水平,或者当使用活细胞时,可以使用细胞数量来标准化数据。
  4. 数据可以表示为在设定时间内的速率(图2A)或通过显示动力学曲线(图2B);也见2016年Larson-Casey等人。


    图2.表达空的或组成型活性的Akt1(Akt1 CA)表达载体的巨噬细胞中的线粒体H 2 O 2 O 2代 >

笔记

  1. 该测定通常在新鲜分离的线粒体(参见方案配方)或膜系统上进行
  2. 在使用当天准备所有工作溶液,不要重复使用。

食谱

  1. pHPA缓冲液
    在Ca 2+ 2+/Mg 2+ 2+ /无酚红的HBSS中制备的总体积为50ml。
    在50 ml锥形管中补充缓冲液,避光



  2. 1mM H 2 O 2工作原液溶液
    1.13μl30%(w/w)H 2 2 O 2储备溶液/10ml H 2 O 2 / 注意:H > MW = 84.01,30%(w/w)(H 2 O em> stock solution)相当于8.82M。
  3. 线粒体缓冲液
    10mM Tris,pH7.8 0.2 mM EDTA
    320mM蔗糖 1蛋白酶抑制剂片剂
    磷酸酶抑制剂稀释1:100

致谢

这项工作由2R01ES015981& VA优点评论BX001135。该协议最初是从Panus等人修改的 。 (1993)。

参考文献

  1. He,C.,Murthy,S.,McCormick,ML,Spitz,DR,Ryan,AJ and Carter,AB(2011)。  巨噬细胞Akt1激酶介导的线粒体调节凋亡抵抗和肺纤维化。 44(3):582-596。 >
  2. Larson-Casey,JL,Murthy,S.,Ryan,AJ和Carter,AB(2014)。  通过Akt调节甲羟戊酸途径调节巨噬细胞存活和肺纤维化的发展。 J Biol Chem 289(52):36204-36219。 br />
  3. Murthy,S.,Ryan,A.,He,C.,Mallampalli,RK和Carter,AB(2010)。  检测从血管内皮细胞释放的H 2 O 2 /em> 14(2):217-223。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Larson-Casey, J. L. and Carter, A. (2016). Determination of H2O2 Generation by pHPA Assay. Bio-protocol 6(22): e2010. DOI: 10.21769/BioProtoc.2010.
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