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Hydrogen Peroxide Measurement in Arabidopsis Root Tissue Using Amplex Red
Amplex Red荧光法测定拟南芥根组织中的过氧化氢酶   

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参见作者原研究论文

本实验方案简略版
Plant Physiology
Jan 2016

Abstract

This protocol describes the measurement of hydrogen peroxide (H2O2) content in Arabidopsis root tissue by using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. When root tissue is disrupted and resuspended in phosphate buffer, H2O2 is released from the cells. The obtained root extracts containing H2O2 can be mixed with a solution containing Amplex® Red reagent (10-acetyl-3,7-dihydrophenoxazine). In the presence of horseradish peroxidase, the Amplex® Red reagent reacts with H2O2 in a 1:1 stoichiometry. The resulting product is the red-fluorescent compound resorufin which can be detected fluorometrically or spectrophotometrically. Our protocol is based on the manual of the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit and describes a step-by-step procedure with a detailed description of the necessary controls and data analysis. We have also included modifications of the protocol, notes and examples that intend to aid the user in easily reproducing the assay with their own samples.

Background

Reactive oxygen species (ROS), such as H2O2, can be generated in the cell as a result of a developmental cue or a stress condition. In high amounts, ROS accumulation can be detrimental by causing cellular damage. However, increased ROS production can also have a signaling role and serve as a secondary messenger in controlling downstream cellular responses. In plants, a signaling role for ROS has been shown for many abiotic stresses, such as drought, salinity, temperature stress, and nutrient deprivation (Mittler, 2002; Mittler and Blumwald, 2015; Xia et al., 2015). In our recent publication Le et al. (2016), we have investigated the connection between ROS production and iron (Fe) deficiency response regulation by investigating the H2O2 content of roots from wild type and mutant Arabidopsis plant lines grown under sufficient and deficient Fe supply conditions.

Materials and Reagents

  1. Kimtech® science precision tissues (Carl Roth, catalog number: AA63.1 )
  2. 2 ml microcentrifuge tubes, safe-seal (SARSTEDT, catalog number: 72.695.500 )
  3. Pipet tips  
  4. Aluminium foil
  5. 96-well microtiter plates for absorbance measurement (e.g., UV-Star® microplate, 96 well, half area, µClear® [Greiner Bio-One, catalog number: 675801 ])
  6. 96-well microtiter plates for fluorescence measurement (e.g., 96 well, half area, black [Greiner Bio-One, catalog number: 675076 ])
  7. Arabidopsis seeds
  8. Sterile distilled water
  9. Plant agar (Duchefa Biochemie, catalog number: P1001.1000 )
  10. Liquid nitrogen
  11. Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A22188 )
  12. H2O2 working solution
  13. HRP enzyme
  14. Horseradish peroxidase
  15. Sodium hypochlorite (NaOCl) (Carl Roth, catalog number: 9062.3 )
  16. Triton X-100 (SERVA Electrophoresis, catalog number: 37238 or Sigma-Aldrich, catalog number: X-100 )
  17. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M9397 )
    Note: This product has been discontinued. Alternatively, MgSO4·7H2O from Carl Roth can be used (Carl Roth, catalog number: T888 )
  18. Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P0662 ; or Carl Roth, catalog number: 3904 )
  19. Potassium nitrate (KNO3) (Carl Roth, catalog number: P021.2 )
  20. Calcium nitrate tetrahydrate [Ca(NO3)2·4H2O] (Carl Roth, catalog number: P740.2 )
  21. Potassium chloride (KCl) (Carl Roth, catalog number: 6781.1 )
  22. Boric acid (H3BO3) (Carl Roth, catalog number: 6943.1 )
  23. Manganese sulfate (MnSO4) (AppliChem, catalog number: A1038 ; or Carl Roth, catalog number: X890.1 )
  24. Zinc sulfate heptahydrate (ZnSO4·7H2O) (VWR, catalog number: VWRC29253.236 )
  25. Copper sulfate pentahydrate (CuSO4·5H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: AC197720050 )
  26. Ammonium heptamolybdate tetrahydrate [(NH4)6Mo7O24·4H2O] (Sigma-Aldrich, catalog number: 431346 ; or Carl Roth, catalog number: 7311 )
  27. D(+)-sucrose (Carl Roth, catalog number: 4621.1 )
  28. Ferric sodium ethylenediaminetetraacetic acid (FeNaEDTA) (Carl Roth, catalog number: 8043.1 )
  29. 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-4’,4’’-disulfonic acid sodium salt (Ferrozine) (Sigma-Aldrich, catalog number: 82950 )
  30. Sterilization solution (see Recipes)
  31. Hoagland medium (see Recipes)
  32. Phosphate buffer (see Recipes)

Equipment

  1. Tube rotator (e.g., VWR, catalog number: 10136-084 )
  2. Mortars and pestles (e.g., MTC Haldenwanger)
  3. Centrifuge
  4. Multi-channel pipet
  5. Analytical scales, e.g., ALJ160_4NM (Kern)
  6. Benchtop centrifuge with cooling (e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Heraeus Fresco 21 Centrifuge )
  7. Plate reader (e.g., Tecan Trading, model: Infinite® M200 Pro )
    Note: The plate reader should be able to excite in the range of 530-560 nm and detect fluorescence at approx. 590 nm, or detect absorbance at approx. 560 nm.
  8. Homogenizer (e.g., such as Precellys® 24 homogenizer) (VWR, catalog number: 432-3750 )

Software

  1. Microsoft Excel

Procedure

A graphical overview of the different protocol steps starting with sample preparation and finishing with resorufin detection is provided in Figure 1.



Figure 1. A graphical overview of the different protocol steps for H2O2 measurement describing the preparation of samples, H2O2 standards and negative controls, which are color coded depending on their identity. The same color code is used for the plate setup in Figure 3.

  1. Plant extract preparation for H2O2 content measurement
    1. Grow Arabidopsis plants to the desired age under the desired growth conditions.
      1. In our case, Arabidopsis seeds were surface sterilized as described previously (Lingam et al., 2011). The seeds were incubated for 8 min in sterilization solution (see Recipes) at room temperature on a tube rotator and subsequently washed five times with sterile distilled water. After the fifth wash, the seeds were stored in 0.1% plant agar for 1-2 days in the dark at 4 °C for stratification.
        Note: Do not incubate the seeds in the sterilization solution for longer than 8 min since this can lead to a reduced seed viability.
      2. Seeds were plated out and germinated on upright Hoagland medium agar plates (see Recipes). Seedlings were grown under long-day conditions (16 h light/21 °C and 8 h dark/19 °C) for 5, 7, 8 and 10 days before harvesting (an example of plates with 10-day-old seedlings is shown in Figure 2). Roots were separated from the shoot directly on the agar plate on which the plants were growing (in order to avoid drying of the roots) and quickly collected into a bunch. The bunch of roots was then very briefly (for a second) and gently laid on a Kimtech precision paper to remove excess moisture before freezing it in liquid nitrogen.


        Figure 2. Example of wild-type Arabidopsis plants (Col-0) grown on upright Hoagland medium agar plates for 10 days. The plants were either grown continuously on sufficient (left) or deficient (right) Fe supply. Scale bars = 1 cm.

    2. Grind frozen roots with mortar and pestle in liquid nitrogen.
    3. Collect the frozen tissue powder in pre-cooled 2 ml microcentrifuge tubes and weigh the samples on analytical scales.
    4. Add 200 µl of ice-cold phosphate buffer (see Recipes) to 30 mg of tissue powder and resuspend on ice. If the amount of powder differs adjust the buffer volume accordingly. For example, for 15 mg powder add 100 µl of phosphate buffer. In order to avoid plant material loss due to sticking to pipet tips we do not resuspend the powder by pipetting up and down. Rather, we let the samples on ice, with the added buffer, for not more than a minute under the powder is well soaked. Afterwards, we flip the microcentrifuge tubes until the suspension appears homogenous.
      Note: The minimal amount of root powder that we have been able to use successfully has been 15 mg, which in our growth conditions corresponded to approx. 25 10-day-old seedlings.
    5. Centrifuge for 3 min at 16,200 x g (corresponds to 13,000 rpm in the centrifuge suggested in Equipment), 4 °C. Transfer the supernatant to a new pre-cooled microcentrifuge tube. This is the sample that will be used for the H2O2 assay (see Figure 3A for an example of sample identity). Keep the samples on ice and proceed, at best, immediately with the H2O2 measurement. We do not have data on how stable the H2O2 in the obtained extracts is.

  2. H2O2 measurement
    1. Prepare 10 mM Amplex® Red reagent stock solution, 1x reaction buffer (0.05 M sodium phosphate buffer, pH 7.4), 10 U/ml horseradish peroxidase (HRP) stock solution, and 20 mM H2O2 working solution according to the instructions provided with the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit.
      Notes:
      1. The Amplex® Red reagent is air and light sensitive. The 1x reaction buffer is stable at room temperature. The HRP stock solution should be aliquoted and kept at -20 °C. The 20 mM H2O2 working solution is stable only for a few hours and should, therefore, be prepared fresh every time.
      2. The manufacturer’s protocol of the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit recommends to perform the H2O2 assay in a 100 µl total volume per reaction. We were able to successfully perform the assay with half the volume (50 µl). Therefore, the following protocol is based on a 50 µl reaction volume.
    2. Prepare an H2O2 standard curve by diluting the respective amount of 20 mM H2O2 working solution with 1x reaction buffer to obtain H2O2 standards of 0 to 10 µM, in 25 µl each. The final H2O2 concentration per reaction will be two-fold lower (e.g., 0 to 5 µM).
      Notes:
      1. The 0 µM H2O2 standard is one of the important negative controls that shows the background fluorescence coming from the mixture of Amplex® Red reagent and HRP in 1x reaction buffer in the absence of H2O2 (see negative control ‘No H2O2’ in Figure 3B).
      2. In our case, we used standards with the following final concentration – 0, 1, 2, 3, 4, and 5 µM H2O2 (see the loading scheme in Figure 3C).
    3. Prepare additional negative controls. A negative control ‘Amplex® Red background’ (see control A in Figure 3B) contains only Amplex® Red reagent in 1x reaction buffer and is needed in order to monitor the background fluorescence/absorbance of the Amplex® Red reagent itself. Another negative control, termed here ‘Sample background’ (see control C in Figure 3B) is prepared for each H2O2 extract and contains the extract in 1x reaction buffer together with the HRP enzyme but without Amplex® Red reagent. This control allows to monitor the individual sample background that could come from the presence of plant compounds that could potentially serve as substrates for HRP, mimicking resorufin production.
    4. Pipet 25 µl of the H2O2 standards, the controls, and the samples (from step A5) into a microtiter plate.
      Note: In our case, we did not have to dilute the samples with 1x reaction buffer prior to the assay. However, it may be necessary to prepare serial dilutions of the sample in order to determine the optimal sample amount per assay. As stated in the manual of the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit, high H2O2 levels may lead to lower fluorescence being detected due to the ability of the excess H2O2 to oxidize resorufin (the fluorescent reaction product) to the nonfluorescent resazurin.
    5. Prepare a working solution of 100 µM Amplex® Red reagent and 0.2 U/ml horseradish peroxidase using the solutions from step B1 (see Table 1), in a light-protected tube (e.g., wrapped in aluminum foil).

      Table 1. Working solution composition. Exemplary calculation for 2.5 ml solution, sufficient for approx. 100 assays.



      Figure 3. An example of samples, necessary controls and standards, and a plate setup. A. An example of sample identity. In this case, wild-type Arabidopsis plants (WT) were grown for 10 days on sufficient (+Fe) or deficient (-Fe) supply in three biological replicates (1 to 3). The H2O2-containing root extracts from these plants were used for the negative controls ‘Sample background’ (C1 to C6) and as samples (S1 to S6). B. A composition summary of the necessary negative controls, samples and standards. C. An example of a plate setup in a 96-well microtiter plate. Each control, sample or standard was pipetted in two technical replicates.


      Figure 4. Example settings for the plate reader Infinite® M200 Pro for measuring resorufin fluorescence (A) and absorbance (B). Note that the Z-Position (optimal measuring height in a well) is calculated based on a well that is expected to contain a high amount of H2O2 and serves as a positive control. In this case, this is well E6 containing one of the two technical replicates of the 5 µM H2O2 standard as shown in the plate setup on Figure 3C.

    6. Begin the reaction by adding 25 µl of working solution to each microtiter-plate well containing the H2O2 standards, the controls, and the samples (pipetted in advance in step B4). See also Note 4.
    7. Incubate the reactions at room temperature for 30 min in the dark.
      Note: Since the assay is continuous, the reaction kinetics can be followed by performing measurements at several time points.
    8. Detect resorufin production using a plate reader by measuring either the fluorescence (excitation at 530-560 nm, emission at 590 nm) or the absorbance at 560 nm (see Figure 4 for example settings). See Note 5.

Data analysis

We suggest to perform the H2O2 measurement on samples from three biological repetitions with two technical replicates each. Here, a technical replicate means two independent enzymatic reactions using the same control, standard or root extract.
An example of raw data, H2O2 standard curve and data analysis representation for a fluorescence measurement on root H2O2 extracts from 10 day-old seedlings is shown in Figure 5.

  1. Calculations based on fluorescence measurements
    1. Calculate the mean of the two technical replicates for the negative control ‘Amplex® Red background’, i.e., Mean (control A) (see also Figures 3B and 3C) and the mean of the two technical replicates for the H2O2 standards, i.e., Mean (Std).
    2. Subtract the Mean (control A) value from Mean (Std) value, i.e., Mean (Std-A).
    3. Correct for background fluorescence coming from the working solution components by subtracting the negative control ‘No H2O2’ (abbreviated as Std 0 in Figures 3B and 3C) from the Mean (Std-A) values for each H2O2 standard, including the Std 0 itself, so that the value for Std 0 becomes 0.
    4. Plot the values obtained in step A3 against the final H2O2 concentration of each standard. Obtain the equation of the trend line (see Figure 5B for an example).
    5. Calculate the mean fluorescence of the two technical replicates for each sample, termed Mean (Sample), and each negative control ‘Sample background’ (abbreviated as C in Figures 3B and C), termed Mean (control C).
    6. Correct for background fluorescence coming from the plant extract itself by subtracting the Mean (control C) values from the Mean (Sample) values, obtaining Mean (Sample-C).
    7. Calculate the H2O2 concentration (in µM) for each sample by using the equation obtained from the H2O2 standard curve in step A4.
    8. As described in the Procedure section, step A4, 200 µl phosphate buffer are added to 30 mg of tissue powder, and this ratio between tissue and buffer volume is kept throughout all samples. Therefore, 7.67 µl contain 1 mg of tissue [(200+30)/30]. Thus, 25 µl of root extract are equivalent to 3.26 mg of root material.
    9. The values obtained in step A7 represent the H2O2 concentration in µM (i.e., pmol/µl). Since the measurement is performed in 50 µl, multiply the number of H2O2 pmol per µl by 50 and divide this by 3.26 mg (see step A8) in order to normalize the measurement to the amount of root material per reaction. The obtained values represent the H2O2 content, e.g., in pmol H2O2/mg FW (FW-fresh weight).
    10. Calculate the mean H2O2 content of the three biological replicates of each line and growth condition, using the values obtained in step A9. Calculate standard deviation.
    11. Use the mean H2O2 content and standard deviation from step A10 for the final presentation of the data (see Figure 5C for an example).


      Figure 5. An example of raw data, H2O2 standard curve, and data analysis representation from a fluorescence measurement on root H2O2 extracts of 10 day-old seedlings. A. An example of raw data. The corresponding plate setup is shown in Figure 3C. B. H2O2 standard curve generated from the raw data in (A). C. An example of data analysis representation.

  2. Calculations based on absorbance (Abs) measurements
    1. The calculation of H2O2 content based on absorbance is performed in the same way as for the fluorescence measurement. The only difference is that here two wavelengths are used – 560 nm for the measurement and 650 nm as a reference wavelength. Therefore, for the calculations, the difference between the Abs(560) and the Abs(650) values has to be used.

Notes

  1. Based on our experience, it is very important that the components of the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit are as fresh as possible and that they are protected from light, air and repeated freeze-thaw cycles. Suboptimal component quality results in bigger variations between the technical and biological replicates.
  2. Another source of variability comes from the root weight measurement. Here, it is very important to collect and freeze roots that are equally dried on paper before grinding them and determining their weight (as described in ‘Procedure’ section A1b).
  3. For high-throughput experiments, it may be advantageous to use a tissue homogenizer (e.g., such as Precellys® 24 homogenizer). In this case, instead of steps A2 and A3 of the ‘Procedure’ section, weigh the roots in a microcentrifuge tube, record the exact weight (e.g., ~30 mg) and grind the roots with the tissue homogenizer. Then, add the corresponding amount of ice-cold phosphate buffer, resuspend and centrifuge, as described under ‘Procedure’, steps A4 and A5.
  4. Since the assay is continuous, care should be taken to minimize the time difference between the first and last sample when pipetting the working solution to the samples, standards and negative controls (as described in ‘Procedure’ B6).Our preferred way is to use a multi-channel pipet when adding the working solution. In this way, the pipetting and measuring times are very similar (since the microtiter plate reader that we are using is measuring well by well).
  5. Often using the fluorescence measurement for calculating the H2O2 content gives less variation between the replicates, resulting in lower standard deviations and clearer differences between the different genotypes and growth conditions. However, it is advisable each time to measure both fluorescence and absorbance.

Recipes

  1. Sterilization solution
    6% NaOCl
    0.1% Triton X-100
  2. Hoagland medium
    0.75 mM MgSO4
    0.5 mM KH2PO4
    1.25 mM KNO3
    1.5 mM Ca(NO3)2
    50 µM KCl
    50 µM H3BO3
    10 µM MnSO4
    2 µM ZnSO4
    1.5 µM CuSO4
    0.075 µM (NH4)6Mo7O24
    1% sucrose (pH 5.8)
    1.4% plant agar
    The medium was supplemented with 50 µM FeNaEDTA (sufficient Fe supply) or 0 Fe, 50 µM ferrozine (deficient Fe supply)
  3. Phosphate buffer
    20 mM K2HPO4 (pH 6.5)

Acknowledgments

This protocol was adapted from our published work (Le et al., 2016) and the manual of the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (https://tools.thermofisher.com/content/sfs/manuals/ mp22188.pdf). This work was supported by the Heinrich-Heine University, Düsseldorf, Germany.

References

  1. Le, C. T., Brumbarova, T., Ivanov, R., Stoof, C., Weber, E., Mohrbacher, J., Fink-Straube, C. and Bauer, P. (2016). ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) linking iron deficiency and oxidative stress responses. Plant Physiol 170(1): 540-557.
  2. Lingam S1, Mohrbacher J, Brumbarova T, Potuschak T, Fink-Straube C, Blondet E, Genschik P, Bauer P. (2011). Interaction between the bHLH transcription factor FIT and ETHYLENE INSENSITIVE3/ETHYLENE INSENSITIVE3-LIKE1 reveals molecular linkage between the regulation of iron acquisition and ethylene signaling in Arabidopsis. Plant Cell 23(5): 1815-1829
  3. Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7(9): 405-410.
  4. Mittler, R. and Blumwald, E. (2015). The roles of ROS and ABA in systemic acquired acclimation. Plant Cell 27(1): 64-70.
  5. Xia, X. J., Zhou, Y. H., Shi, K., Zhou, J., Foyer, C. H. and Yu, J. Q. (2015). Interplay between reactive oxygen species and hormones in the control of plant development and stress tolerance. J Exp Bot 66(10): 2839-2856.

简介

该方案描述了在拟南芥根组织中通过使用Amplex来测量过氧化氢(H 2 O 2 O 2)含量。 sup>红色过氧化氢/过氧化物酶测定试剂盒。当根组织被破坏并重新悬浮在磷酸盐缓冲液中时,H 2 O 2 O 2从细胞中释放。可以将获得的含有H 2 O 2 O 2的根提取物与含有Amplex Red试剂(10-乙酰基-3,7-二 - 。在辣根过氧化物酶的存在下,Amplex Red试剂与H 2 O 2以1:1的化学计量反应。所得产物是红色荧光化合物试卤灵,其可以通过荧光测定或分光光度法检测。我们的方案是基于Amplex ®红色过氧化氢/过氧化物酶测定试剂盒的手册,并描述了一步一步的程序,详细描述必要的控制和数据分析。我们还包括旨在帮助用户容易地用其自己的样品再现测定的方案,说明和实施例的修改。

[背景] 活性氧(ROS),例如H 2 O 2 O 2可以在该细胞是发育提示或应激条件的结果。在大量,ROS积累可能是有害的,通过造成细胞损伤。然而,增加的ROS产生也可以具有信号传导作用,并且作为控制下游细胞反应的第二信使。在植物中,对于许多非生物胁迫,例如干旱,盐度,温度胁迫和营养缺乏,已经显示ROS的信号传导作用(Mittler,2002; Mittler和Blumwald,2015; Xia等人)。 ,2015)。在我们最近的出版物Le em等人中。 (2016)中,我们通过研究来自野生型和突变体的根的H 2 O 2亚型含量,研究了ROS产生和铁(Fe)缺乏反应调节之间的关系。 em>拟南芥植物品系在足够和缺乏的Fe供应条件下生长。

材料和试剂

  1. Kimtech ?科学精密组织(Carl Roth,目录号:AA63.1)
  2. 2ml微量离心管,安全密封(SARSTEDT,目录号:72.695.500)
  3. 吸头提示
  4. 铝箔
  5. 用于吸光度测量的96孔微量滴定板(例如 UV-Star 微孔板,96孔,半面积,μClear Bio-One,目录号:675801])
  6. 用于荧光测量的96孔微量滴定板(例如96孔,半面积,黑色[Greiner Bio-One,目录号:675076])
  7. 拟南芥种子
  8. 无菌蒸馏水
  9. 植物琼脂(Duchefa Biochemie,目录号:P1001.1000)
  10. 液氮
  11. 红色过氧化氢/过氧化物酶测定试剂盒(Thermo Fisher Scientific,Molecular Probes TM ,目录号:A22188)
  12. H <2>工作溶液
  13. HRP酶
  14. 辣根过氧化物酶
  15. 次氯酸钠(NaOCl)(Carl Roth,目录号:9062.3)
  16. Triton X-100(SERVA Electrophoresis,目录号:37238或Sigma-Aldrich,目录号:X-100)
  17. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M9397)
    注意:此产品已停产。或者,MgSO 可以使用Carl Roth(Carl Roth,目录号:T888)。
  18. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P0662;或Carl Roth,目录号:3904)
  19. 硝酸钾(KNO 3)(Carl Roth,目录号:P021.2)
  20. 硝酸钙四水合物[Ca(NO 3)2·4H 2 O](Carl Roth,目录号:P740.2)
  21. 氯化钾(KCl)(Carl Roth,目录号:6781.1)
  22. 硼酸(H 3 BO 3)(Carl Roth,目录号:6943.1)
  23. 硫酸锰(MnSO 4)(AppliChem,目录号:A1038;或Carl Roth,目录号:X890.1)
  24. 硫酸锌七水合物(ZnSO 4·7H 2 O)(VWR,目录号:VWRC29253.236)
  25. 硫酸铜五水合物(CuSO 4·5H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:AC197720050)
  26. 七钼酸铵四水合物[(NH 4)6 Mo 12 Mo 24 SO 4·4H 2 > O](Sigma-Aldrich,目录号:431346;或Carl Roth,目录号:7311)
  27. D(+) - 蔗糖(Carl Roth,目录号:4621.1)
  28. 乙二胺四乙酸铁(FeNaEDTA)(Carl Roth,目录号:8043.1)
  29. 3-(2-吡啶基)-5,6-二苯基-1,2,4-三嗪-4',4" - 二磺酸钠盐(Ferrozine)(Sigma-Aldrich,目录号:82950)
  30. 灭菌溶液(参见配方)
  31. Hoagland培养基(参见食谱)
  32. 磷酸盐缓冲液(参见配方)

设备

  1. 管旋转器(例如。,VWR,目录号:10136-084)
  2. 迫击炮和杵(,例如MTC Haldenwanger)
  3. 离心机
  4. 多通道移液器
  5. 分析标度,例如。,ALJ160_4NM(Kern)
  6. 带有冷却的台式离心机(例如Thermo Fisher Scientific,Thermo Scientific TM ,型号:Heraeus Fresco 21离心机)
  7. 读板器(例如,Tecan Trading,型号:Infinite M200 Pro)
    注意:酶标仪应该能够在530-560nm范围内激发, 590nm,或检测约50nm的吸光度。 560 nm。
  8. 均化器(例如,例如Precellys 24均化器)(VWR,目录号:432-3750)

软件

  1. Microsoft Excel

程序

图1提供了从样品制备开始并使用试卤灵检测完成的不同方案步骤的图形概述



图1.描述样品H 2 O 2的测定的不同协议步骤的图形概述,其中H <2> 2 标准和阴性对照,它们根据其身份进行了颜色编码。图3中的板设置使用相同的颜色代码。

  1. 用于H 2 O 2含量测量的植物提取物制备
    1. 在所需的生长条件下将拟南芥植物生长至所需的年龄。
      1. 在我们的情况下,如先前所述(Lingam等人,2011),将拟南芥种子表面灭菌。将种子在灭菌溶液(参见Recipes)中在室温下在管旋转器上温育8分钟,随后用无菌蒸馏水洗涤5次。第五次洗涤后,将种子在4℃下在黑暗中储存在0.1%植物琼脂中1-2天以进行分层。
        注意:不要将种子在灭菌溶液中孵育超过8分钟,因为这会导致种子生存力降低。
      2. 将种子播种并在直立的Hoagland培养基琼脂平板上发芽(参见Recipes)。幼苗在长日照条件下(16小时光/21℃和8小时黑暗/19℃)生长5,7,8和10天,然后收获(显示具有10天龄幼苗的平板的实例在图2中)。在植物生长的琼脂平板上直接从枝上分离根(以避免根的干燥),并迅速收集成束。然后非常短暂(一秒钟),轻轻地放在Kimtech精密纸上,以除去多余的水分,然后将其冷冻在液氮中。


        图2.在直立的Hoagland培养基琼脂平板上生长10天的野生型拟南芥植物(Col-0)的实例。将植物连续生长足够(左侧)或缺乏(右)铁供应。比例尺= 1厘米。

    2. 用液体氮气中的研钵和杵研磨冷冻的根
    3. 在预冷的2ml微量离心管中收集冷冻的组织粉末,并在分析秤上称重样品
    4. 加入200微升冰冷的磷酸盐缓冲液(见配方)30毫克的组织粉末,并重悬在冰上。如果粉末量不同,则相应地调整缓冲体积。例如,对于15mg粉末,加入100μl磷酸盐缓冲液。为了避免由于粘在移液管吸头上造成植物材料损失,我们不会通过上下移液来重悬粉末。相反,我们让冰上的样品与添加的缓冲液在粉末下不超过一分钟被良好地浸泡。然后,我们翻转微量离心管直到悬浮液看起来均匀 注意:我们已经能够成功使用的根粉的最小量已经是15mg,其在我们的生长条件对应于约。 25 10日龄的幼苗。
    5. 在16,200×g离心3分钟(对应于在设备中建议的离心机中13,000rpm),4℃。将上清液转移到新的预冷却的微量离心管。这是将用于H 2 O 2 O 2分析的样品(对于样品同一性的实例参见图3A)。将样品保持在冰上,并最多立即进行H 2 O 2 O 2测量。我们没有关于所获得的提取物中H 2 O 2 O 2的稳定性的数据。

  2. H 2 测量
    1. 制备10mM Amplex Red试剂储备溶液,1×反应缓冲液(0.05M磷酸钠缓冲液,pH 7.4),10U/ml辣根过氧化物酶(HRP)储备溶液和20mM H 2 SO 4,根据Amplex的过氧化氢/过氧化物酶测定试剂盒提供的说明书,测定2 H 2 O 2工作溶液。
      注意:
      1. 红色试剂是空气和光敏感的。 1x反应缓冲液在室温下是稳定的。应将HRP储备溶液等分并保存在 - 0°C。 2 em> 2 工作溶液只有几个小时才能稳定,因此每次都应准备新鲜。
      2. < em>< em>红色过氧化氢/过氧化物酶测定试剂盒的制造商方案推荐进行H 2 我们能够成功地执行半体积(50μl)的测定。因此,以下方案基于50μl反应体积。
    2. 通过稀释各自量的20mM H 2 O 2 O 2工作溶液制备H 2 O 2 O 2标准曲线与1×反应缓冲液混合以获得每种25μl的H 2 O 2 O 2标准物(0至10μM)。每个反应的最终H 2 O 2 O 2浓度将低两倍(例如,0至5μM)。
      注意:
      1. 0μMH 2 em>标准是重要的阴性对照之一,其显示来自Amplex 红色试剂和HRP在1x反应中的混合物的背景荧光在没有H 2 >(见阴性对照'否H 2 ')。
      2. 在我们的情况下,我们使用具有以下最终浓度的标准物 - 0,1, 2 ,3,4和5μMH 2 (见图3C中的加载方案)/em>
    3. 准备其他阴性对照。阴性对照'Amplex 红色背景'(参见图3B中的对照A)在1x反应缓冲液中仅含有Amplex Red试剂,并且为了监测背景Amplex Red试剂本身的荧光/吸光度。为每个H 2 O 2 Sub提取物制备另一个阴性对照,在此称为"样品背景"(参见图3B中的对照C),并且将提取物在1x反应缓冲液中一起与HRP酶,但没有Amplex Red试剂。该对照允许监测可能来自可能用作HRP的底物的植物化合物的存在的单个样品背景,模拟试卤灵的生产。
    4. 将25μlH 2 O 2 Sub 2标准品,对照和样品(来自步骤A5)吸移到微量滴定板中。
      注意:在我们的情况下,我们不需要在测定前用1x反应缓冲液稀释样品。然而,可能有必要制备样品的系列稀释物,以确定每次测定的最佳样品量。如Amplex 红色过氧化氢/过氧化物酶测定试剂盒手册中所述,高H 2 荧光反应产物)转化为非荧光刃天青。
    5. 使用来自步骤B1(参见表1)的溶液,在避光管中(例如,1小时)制备100μMAmplex Red试剂和0.2U/ml辣根过氧化物酶的工作溶液, ,用铝箔包裹)。

      表1.工作溶液组成。2.5 ml溶液的示例计算,足够大约。 100分析


      图3.样品,必要的对照和标准品以及样品板设置示例。A.样品标识示例。在这种情况下,野生型拟南芥植物(WT)在三个生物重复(1-3)中在足够(+ Fe)或缺乏(-Fe)的供应下生长10天。将来自这些植物的含有H 2 O 2 O 2的根提取物用于阴性对照"样品背景"(C1至C6)和样品(S1至S6)。 B.必要的阴性对照,样品和标准的组成概述。 C.在96孔微量滴定板中设置板的实例。每个对照,样品或标准品用两个技术重复移液

      图4.平板读数器的示例设置Infinite ? M200 Pro用于测量试卤灵荧光(A)和吸光度(B)。注意,Z位置(最佳测量高度在井中)基于预期含有高量H 2 O 2 O 2的孔并用作阳性对照的孔来计算。在这种情况下,这是良好的E6,其含有5μMH Sub 2 O 2 2标准的两个技术重复之一,如图3C中的板设置所示。

    6. 通过将25μl工作溶液加入含有H 2 O 2 Sub标准物的每个微量滴定板孔,对照和样品(预先在步骤B4)。另见注4
    7. 在室温下在黑暗中孵育反应30分钟。
      注意:由于测定是连续的,反应动力学可以在几个时间点进行测量。
    8. 通过测量荧光(在530-560nm激发,在590nm发射)或在560nm处的吸光度(参见图4的示例设置),使用读板器检测试卤灵的产生。见注5.

数据分析

我们建议对来自三个生物重复的样品进行H 2 O 2 O 2的测量,其中每个具有两个技术重复。这里,技术重复是指使用相同对照,标准或根提取物的两个独立的酶反应 原始数据的实例,H 2 O 2 O 2标准曲线和用于在根H 2 O 2上的荧光测量的数据分析表示来自10日龄幼苗的提取物显示于图5中。

  1. 基于荧光测量的计算
    1. 计算阴性对照"Amplex Red background",即,Mean(对照A)(也参见图3B和3C)的两个技术重复的平均值,对于H sub 2 O 2 sub标准的两个技术重复的平均值,即平均值(std)。
    2. 从Mean(Std)值中减去Mean(对照A)值,即。,Mean(Std-A)。
    3. 通过从平均值减去阴性对照'No H sub 2 O 2'(在图3B和3C中缩写为Std 0),校正来自工作溶液组分的背景荧光(Std-A)值,包括Std 0本身,使得Std 0的值变为0.
      (对于每个H sub 2 O 2 <
    4. 将步骤A3中获得的值相对于每种标准品的最终H 2 O 2 O 2浓度绘图。获取趋势线的方程(参见图5B的示例)。
    5. 计算称为平均值(样品)的每个样品的两个技术重复的平均荧光,以及称为平均值(对照C)的每个阴性对照'样品背景'(在图3B和C中缩写为C)。
    6. 通过从平均值(样品)值减去平均值(对照C)值,获得平均值(样品C),校正来自植物提取物本身的背景荧光。
    7. 通过使用从H 2 O 2 O 2浓度获得的方程式计算每个样品的H 2 O 2 O 2浓度(以μM计)/sub>标准曲线。
    8. 如步骤部分,步骤A4中所述,将200μl磷酸盐缓冲液加入到30mg组织粉末中,并且在所有样品中保持组织和缓冲液体积之间的比率。因此,7.67μl含有1mg组织[(200 + 30)/30]。因此,25μl根提取物相当于3.26mg根材料
    9. 在步骤A7中获得的值表示以μM(即,pmol /μl)表示的H 2 O 2 sub浓度。由于测量在50μl中进行,将每μL的H 2 O 2 O 2 pmol的数量乘以50,并除以3.26mg(参见步骤A8),以便将测量标准化为每次反应的根材料的量。获得的值表示在pmol H 2 O 2中的H 2 O 2含量,例如 /mg FW(FW鲜重)。
    10. 使用在步骤A9中获得的值计算每条线的三个生物重复和生长条件的平均H 2 O 2 O 2含量。计算标准偏差。
    11. 对于数据的最终呈现使用来自步骤A10的平均H 2 O 2内容和标准偏差(例如参见图5C)。< br />

      图5.原始数据,H 2 O 2 O 2标准曲线的实例和来自根H 2 O 2上的荧光测量的数据分析表示, sub> O 提取10天龄的幼苗。 A.原始数据的一个例子。相应的板设置如图3C所示。 (A)中的原始数据生成的H 2 H 2 O 2标准曲线。 C.数据分析表示的示例。

  2. 基于吸光度(Abs)测量的计算
    1. 基于吸光度的H 2 O 2含量的计算以与荧光测量相同的方式进行。唯一的区别是,在这里使用两个波长 - 560nm用于测量,650nm用作参考波长。因此,对于计算,必须使用Abs(560)和Abs(650)值之间的差。

笔记

  1. 基于我们的经验,非常重要的是,Amplex 红色过氧化氢/过氧化物酶测定试剂盒的组分尽可能新鲜,并且保护它们免受光,空气和反复冻融循环。次优组件质量导致技术和生物复制品之间的更大变化
  2. 变异性的另一个来源是根重量测量。这里,非常重要的是收集和冻结在纸上同样干燥的根,然后研磨它们并确定它们的重量(如"程序"部分A1b中所述)。
  3. 对于高通量实验,可能有利的是使用组织匀浆器(例如,例如Precellys24匀浆器)。在这种情况下,代替"程序"部分的步骤A2和A3,称重微量离心管中的根,记录精确重量(例如。约30mg),并用组织匀浆器。然后,按照"程序",步骤A4和A5所述,添加相应量的冰冷磷酸盐缓冲液,重悬和离心。
  4. 由于测定是连续的,当吸移工作溶液到样品,标准品和阴性对照(如"程序"B6中所述)时,应注意使第一个和最后一个样品之间的时间差最小化。我们优选的方法是使用添加工作溶液时的多通道移液器。这样,移液和测量时间非常相似(因为我们使用的微量滴定板读数器测量井很好)。
  5. 通常使用荧光测量来计算H 2 O 2 O 2含量在重复之间给出较小的变化,导致不同基因型和生长条件之间较低的标准偏差和更清楚的差异。但是,每次测量荧光和吸光度是可取的

食谱

  1. 灭菌溶液
    6%NaOCl
    0.1%Triton X-100
  2. Hoagland培养基
    0.75mM MgSO 4 0.5mM KH 2 PO 4 sub/
    1.25mM KNO 3
    1.5mM Ca(NO 3)2 sub。 50μMKCl
    50μMH sub 3 BO Sub 3
    10μMMnSO 4
    2μMZnSO 4
    1.5μMCuSO 4
    0.075μM(NH 4)6 Mo 7 Mo 24 SO 4/
    1%蔗糖(pH5.8) 1.4%植物琼脂 培养基补充有50μMFeNaEDTA(足够的Fe供应)或0Fe,50μMferrozine(缺乏Fe供应)
  3. 磷酸盐缓冲液
    20mM K 2 HPO 4(pH 6.5)

致谢

该方案改编自我们公开的工作(Le等人,2016)和Amplex的过氧化氢/过氧化物酶测定试剂盒的手册( https://tools.thermofisher.com/content/sfs/manuals/mp22188 .pdf )。这项工作得到德国杜塞尔多夫海因里希 - 海涅大学的支持。

参考文献

  1. Le,CT,Brumbarova,T.,Ivanov,R.,Stoof,C.,Weber,E.,Mohrbacher,J.,Fink-Straube,C.and Bauer, "AR"> - >与"FER-LIKE铁缺陷诱导转录子"相互作用的"AR-ABOIDIS THALIANA12(ZAT12)"的ZINC指形子"ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26556796"target ="_ blank" FACTOR(FIT)linking iron deficiency and oxidative stress responses。 Plant Physiol 170(1):540-557。
  2. Lingam S1,Mohrbacher J,Brumbarova T,Potuschak T,Fink-Straube C,Blondet E,Genschik P,Bauer P.(2011)。  氧化应激,抗氧化剂和胁迫耐受性。 Trends Plant Sci 7(9):405-410。
  3. Mittler,R。和Blumwald,E。(2015)。  ROS和ABA在系统获得性驯化中的作用。植物细胞27(1):64-70。
  4. als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als als .org/content/66/10/2839.short"target ="_ blank">在植物发育和胁迫耐受的控制中活性氧和激素之间的相互作用。 66(10):2839-2856。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Brumbarova, T., Le, C. T. and Bauer, P. (2016). Hydrogen Peroxide Measurement in Arabidopsis Root Tissue Using Amplex Red. Bio-protocol 6(21): e1999. DOI: 10.21769/BioProtoc.1999.
  2. Le, C. T., Brumbarova, T., Ivanov, R., Stoof, C., Weber, E., Mohrbacher, J., Fink-Straube, C. and Bauer, P. (2016). ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) linking iron deficiency and oxidative stress responses. Plant Physiol 170(1): 540-557.
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