搜索

Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans
秀丽隐杆线虫中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性测定实验方案   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Assays for superoxide dismutase (SOD) and catalase (CAT) activities are widely employed to indicate antioxidant responses underlying the toxic effects of test chemicals. Yet, earlier studies mainly described the procedures as performed according to manufacturer’s instructions without modifications that are specific to any organisms. The present protocol describes the steps in analyzing the superoxide dismutase (SOD) and catalase (CAT) activities in C. elegans, which is a model organism that can be used to study effects of pharmaceutical compounds and environmental pollutants. The main steps include: (1) sample preparation; (2) total protein assay; (3) SOD activity assay; (4) CAT activity assay; and (5) medium list and formula, and also data analysis and performance notes.

Keywords: SOD(SOD), CAT(CAT), Total protein(总蛋白), C. elegans(秀丽隐杆线虫), Protocol(实验方案)

Background

Biomarkers are essential to examine biological and pathogenic processes in response to a chemical, an agent or a therapeutic intervention. Various biological processes in organisms result in reactive oxygen species (ROS) which cause oxidative stress. In response to such oxidative stress, organisms can deploy superoxide dismutase (SOD) and catalase (CAT) to scavenge ROS so as to protect the cellular homeostasis (Balaban et al., 2005). On the one hand, various chemicals (pollutants) can retard such antioxidant responses, and disturb the health of organisms including human beings. On the other hand, many pharmaceuticals aim to strengthen the antioxidant responses to improve health. Therefore, activities of SOD and CAT are very important to reflect potential effects of chemicals or/and pharmaceuticals.

Caenorhabditis elegans (C. elegans) is a model organism that has been used to study effects of pharmaceutical compounds (Dengg and van Meel, 2004; Carretero et al., 2017) and environmental pollutants (Yu et al., 2013a and 2017). Several studies have used SOD and CAT assays to indicate the antioxidant responses and potential mechanism underlying the toxic effects of test chemicals (Feng et al., 2015; Yu et al., 2012; 2016 and 2017). However, these studies simply described that the determination was carried out, by using a generic kit protocol without species-specific modifications. Therefore, the explicit protocols to perform SOD and CAT assays in C. elegans are still needed for better specific instruction.

In the present protocol, we provide a nematode protocol with experimental details to analyze SOD and CAT activities in C. elegans.

Materials and Reagents

  1. Pipette tips (https://online-shop.eppendorf.com)
  2. Plate (60 mm)
  3. Centrifuge tubes, 1.5 ml (Eppendorf, catalog number: 022364111 )
  4. Absorbent paper (KCWW, Kimberly-Clark, catalog number: 0131 )
  5. 96-well plate, with lids (Corning, Costar®, catalog number: 3599 )
  6. Sealing tape, for 96-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 15036 )
  7. Nematodes (wild type N2)
    Note: These nematodes are treated according to each researcher’s experiments. In the present protocol, the nematodes only have difference in numbers.
  8. BCA protein assay kits (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 or 23227 )
    1. Protein standard (5 mg/ml)
    2. BCA reagent A and B
  9. SOD assay kits (Beyotime Biotechnology, catalog number: S0101 )
    1. Reaction initiation solution
    2. SOD detection buffer
    3. WST-8
    4. Enzyme solution
    5. Reaction initiation solution (40x)
  10. CAT assay kits (Beyotime Biotechnology, catalog number: S0051 )
    1. Enzyme conjugate
    2. Wash solution 40x
    3. Substrate A and B
    4. Stop solution
  11. Sodium hydrate (NaOH), Analytic Reagent (Sinopharm Chemical Reagent, catalog number: 10019762 )
  12. Sodium hypochlorite (Antiformin, NaOCl, with 6-14% active Cl), Analytic Reagent (ALADDIN, catalog number: S101636-500 ml )
  13. Potassium phosphate dibasic (K2HPO4), Analytic Reagent (Sinopharm Chemical Reagent, catalog number: 20032118 )
  14. Potassium phosphate monobasic (KH2PO4), Analytic Reagent (Sinopharm Chemical Reagent, catalog number: 10017618 )
  15. Clorox solution (see Recipes)
  16. Phosphate buffered saline/buffer (PBS), pH 7.0 (see Recipes)

Equipment

  1. Pipettes
  2. Microscope
  3. Centrifuge, Eppendorf 5417R (Eppendorf, model: 5417 R , catalog number: 01396)
  4. Pestles, Eppendorf (Eppendorf, catalog number: F0140010 )
  5. Motor-driven tissue grinder (Beijing Baiwan Electronic Technology, catalog number: HG215-LH-A )
  6. Ice bath, in centrifuge tube box
  7. Microplate reader, BioTek (BioTek Instruments, model: Epoch )
  8. Sterilized bottle, Fisherbrand (100 ml, Fisher Scientific, catalog number: FB800100 ; 250 ml, Fisher Scientific, catalog number: FB800250 ; 500 ml, Fisher Scientific, catalog number: FB800500 )
  9. Magnetic stir bar
  10. Magnetic stir plate
  11. pH meter
  12. Incubator, Yiheng (Yiheng, model: LRH-1000F )

Procedure

  1. Sample preparation
    1. Sample collection
      The entire protocol uses the L4 stage nematodes as the reference point. Follow the nematode culture and age-synchronization according to earlier reports (Solis and Petrascheck, 2015; Yu et al., 2017). Wash the L4 stage nematodes off each nematode growth medium plate (60 mm) with 1.6 ml phosphate buffered saline (PBS, pH 7.0) into a 1.5 ml centrifuge tube. After a 30-min settlement by gravity at 4 °C, carefully pipette out and discard the supernatants. Add 500 μl ice-cold PBS, resuspend the pellets with gentle and repetitive pipetting followed by a further 15-min settlement. Then, discard the supernatants, add 500 μl ice-cold PBS, resuspend the pellets with gentle and repetitive pipetting and obtain groups with 20, 50, 100, 200, 300, 400 and 500 nematodes in 200 μl PBS in the centrifuge tubes using the following steps.
      Take the group with 100 nematodes as an example.
      1. Count the nematodes in 20 μl PBS under a microscope with three independent trials to estimate the nematode density in the whole tube.
      2. Based on the number of worms obtained, calculate and adjust the volume of PBS so that the final density is ~9 to 11 worms per 20 μl. For example, if the number is more than 10, add more PBS accordingly; and if the number is less than 10, perform a 10-min settlement and remove corresponding amount of PBS to achieve the desired density.
      3. Transfer 260 μl PBS with the nematodes into new 1.5 ml tubes with pipette, and resuspend the pellets with gentle and repetitive pipetting.
      4. Count nematode numbers in 20 μl PBS in three independent trials, with gentle and repetitive pipetting in between, to confirm the nematode density, leaving 200 μl in the tube. If the nematode density is not consistent with the setting, repeat step A1b.
      5. Prepare groups with other nematode numbers through the same way.
    2. Sample storage
      Centrifuge the tubes at 5,000 x g for 5 min (4 °C). Discard the supernatant PBS carefully with pipette and store the pellets at -26 °C overnight or -80 °C for over a week.
    3. Sample homogenization
      1. Connect a clean pestle with the motor-driven tissue grinder. Wash the pestle in ice-cold PBS, and clean it with the absorbent paper. Push the pestle tightly against the pellets in the 1.5 ml centrifuge tube. Keep the centrifuge tube in an ice bucket (see Figure 1). Start the tissue grinder, grind the pellets for 10 sec with a 2-sec cooling down followed by another 10-sec grinding. Then, use 200 μl ice-cold PBS to wash the residual liquids on the pestle back to the centrifuge tube (see Figure 1), before taking it out of the tube.
      2. Centrifuge the tubes at 5,000 x g for 5 min at 4 °C. Pipette out 200 μl supernatants from each sample. Aliquot them into four tubes with 50 μl in each for subsequent determination.


        Figure 1. Sample homogenization. The left picture shows that motor-driven tissue grinder homogenizes the pellets in the centrifuge tube in an ice bath, and the right one shows that residual liquids are washed off the pestle with 200 μl ice-cold PBS.

  2. Total protein assay
    Measure the total protein (TP) in each sample by enzyme-linked immunosorbent assay (ELISA) kits based on BCA methods.
    1. Preparation for protein standards
      Use the stock protein standard (5 mg/ml) to prepare 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg/ml dilutions, 100 μl each using PBS.
    2. Preparation for BCA working solution
      Prepare BCA working solution with BCA reagent A and B by a volume ratio of 50:1. Total volume of the BCA working solution is calculated according to the amount of tested samples (200 μl per sample).
    3. Determination of protein concentration
      1. For each protein standard, add 20 μl into a 96-well plate with at least two replicates. Use 20 μl PBS buffer as control.
      2. For each nematode sample, add 20 μl from one aliquot into the 96-well plate with at least two replicates. Use up this aliquot to ensure replicate and avoid repetitive freezing-unfreezing of the samples.
      3. Then, add 200 μl BCA working solution per well for all the protein standards and nematode samples. Seal the wells by sealing tape to avoid the evaporation of water, and incubate the plate for 30 min at 37 °C.
      4. Measure the absorbance at 562 nm (A562) using a microplate reader.
    4. Data calculation
      Calculate the protein contents in the samples according to the standard curve.
      One presentation of the relation between the nematode numbers and the protein concentrations is shown in Figure 2.


      Figure 2. The relationship between the nematode numbers and the nematode protein concentrations

  3. SOD activity assay
    Measure the SOD in each sample by ELISA kits based on WST-8 method. The WST-8 method is based on the colorimetric reaction of WST-8. In the reaction, xanthine oxidase (XO) catalyzes the oxidation conversion of xanthine to yield superoxide anion, which quenches WST-8 to produce water-soluble formazan (a purple dye). As SOD quenches superoxide anion, thus its activity inhibits the overall colorimetric reaction (see Figure 3). Therefore, the inhibition levels are used to indicate the SOD activities in cells, tissues or other biological samples.


    Figure 3. The colorimetric reaction of WST-8 with the superoxide anion from xanthine, and the SOD activities inhibit the colorimetric reaction

    1. Preparation of necessary solution
      Prepare the standards and controls, and working solutions according to the manufacturer’s instruction of the ELISA kit.
      1. Each sample needs 160 μl WST-8/enzyme working solution and 20 μl reaction initiation solution. Calculate the total amount according to the number of nematode samples plus standards. For WST-8/enzyme working solution, 160 μl consists of 151 μl SOD detection buffer, 8 μl WST-8 and 1 μl enzyme solution.
      2. Prepare the reaction initiation solution, by diluting from the original stock 1:40 in SOD detection buffer (i.e., 1 μl original solution to 39 μl buffer).
    2. Determination of samples
      1. Add 20 μl standards and controls into a 96-well plate with at least two replicates.
      2. Add 20 μl nematode samples into the 96-well plate with at least two replicates. Notably, each nematode sample is processed and divided into 4 aliquots after the step of sample homogenization. Use up one aliquot to ensure replicate and avoid repetitive freezing-unfreezing of the samples.
      3. Use a multi-channel pipette to add 160 μl WST-8/enzyme working solution into each sample, and then, add 20 μl reaction initiation working solution. The concentrations of standards are 100, 50, 10, 5, 2.5, 1.25 and 0.625 U/ml.
      4. There are two blank controls. For the blank control 1, 20 μl sample are replaced by SOD detection buffer. For the blank control 2, 20 μl sample and 20 μl reaction initiation solution are replaced by 40 μl SOD detection buffer.
      5. Incubate the plate for 30 min at 37 °C.
      6. Read the absorbance at 450 nm (A450) by a microplate reader.
    3. Calculation in SOD activity assay
      1. Inhibition (%) = (ABlank control 1 - AStandard or Sample)/(ABlank control 1 - ABlank control 2) x 100%. Use the standards with known SOD activities and their inhibitions to establish a standard curve. Then, use the curve to calculate the SOD activities of samples by their inhibitions. One example for the standard curve is shown in Figure 4.


        Figure 4. The relationship between SOD activities in logarithm and inhibition (%) on WST-8 colorimetric reaction

      2. Express the SOD enzyme activity in each nematode sample as its proportion (P) in the total protein (TP) of the same sample to eliminate the differences of nematode numbers among samples. The normalized SOD activities in the nematodes are listed in Table 1. The proportion values generally range from 4.04 to 4.35. The values in groups with 20 and 50 nematodes are quite greater than the general range. Although these values are within mean value + 3 standard deviation (SD), we recommend not using them to avoid unreliability. These results indicate that at least 100 nematodes should be used to ensure the feasibility and stability in measuring SOD activities.

        Table 1. Connection between normalized SOD activities and nematode numbers


  4. CAT activity assay
    Measure the CAT activities in each sample with ELISA kits. The assay principle is based on the reaction of catalase to decompose hydrogen peroxide (H2O2). The excess H2O2 can form complexation with ammonium molybdate to produce light yellow solution. The solution’s adsorption at 450 nm can be used to calculate the concentration of H2O2 and therefore to indirectly indicate the CAT activities.
    1. Reagents preparation
      According to the manufacturer’s instruction of the ELISA kit, prepare all reagents before the assay with careful calculation based on the numbers of samples, standards and controls.
    2. Addition of standards or samples
      Add 50 μl standards or samples into a 96-well plate. Use 50 μl PBS as blank controls. All standards, samples and controls are recommended to have at least two replicates. Notably, each nematode sample is processed and divided into 4 aliquots after the step of sample homogenization. Use up one aliquot to ensure replicate and avoid repetitive freezing-unfreezing of the samples. The concentrations of standards are 400, 100, 25, 6.25, 1.5625, 0.4 and 0.1 U/L.
    3. Enzyme conjugation
      Add 100 μl of enzyme conjugate to wells containing standards, samples and blanks. Then, seal the wells by sealing tape to avoid the evaporation of water, and incubate the microplate statically for 60 min at 37 °C.
    4. Wash
      1. Discard the liquid content and tap the assay plate on a piece of absorbent paper to remove residual buffer.
      2. Add 350 μl wash solution (1x, which was diluted from the 40x package with PBS) to each well and keep the microplate static for 1 min. Then, discard the liquid content and tap the assay plate on a piece of absorbent paper to remove residual buffer.
      3. Repeat the above wash process for four times.
    5. Addition of substrates
      Use a multi-channel pipette to add 50 μl substrate A and 50 μl substrate B to each well. Incubate the microplate at 37 °C in the dark for 15 min.
    6. Endpoint measurement
      Add 50 μl stop solution to each well. After 1 min incubation, measure the absorbance at 450 nm on a microplate reader within 15 min.
    7. Data calculation and presentation
      Calculate the total CAT activities of samples according to the standard curve (see Figure 5), the method of which is similar to that used by the SOD activity assay.


      Figure 5. The relationship between total CAT activities in logarithm and inhibition (%) on colorimetric reaction between H2O2 and ammonium molybdate

      Express the CAT activities in each nematode sample as its proportion (P) in the total protein (TP) of the same sample to eliminate the differences of nematode numbers among samples. The normalized CAT activities in the nematodes are listed in Table 2. The proportion values generally range from 8.74 to 10.36. The values in groups with 20 and 50 nematodes are significantly greater than this range. These results indicate that at least 100 nematodes should be used to ensure the feasibility and stability in measuring CAT activities.

      Table 2. Connection between normalized CAT activities and nematode numbers

Data analysis

  1. The data analysis procedure refers to our previous report (Yu et al., 2013b). Firstly, the values of the nematode total protein (TP) in one aliquot of each sample are measured in at least three replicates, and the means of these TP values (TP mean) are calculated to represent the TP concentration in the sample. Secondly, the SOD (or CAT) values in other aliquots of the same sample are also measured in at least three replicates. Thirdly, the SOD (or CAT) values in each replicate are calculated as their proportions to the TP mean value to obtain SOD (or CAT) proportion values. Fourthly, the SOD (or CAT) proportion values are calculated to obtain the mean SOD (or CAT) proportion in the sample and corresponding standard error.
  2. In cases that the samples come from different treatments, the mean SOD (or CAT) proportion in samples from the control are normalized to represent 100% (or 1.0); then, the SOD (or CAT) proportions in samples from the chemical exposure are transformed to percentages of the controls (POCs) (or fold changes against control); next, the POC values (or fold-changes) in the samples are used to calculate the mean POC and corresponding standard error.
  3. The nematode samples should be collected from at least three independently repeated experiments, with one set of data in each experiment. The ANOVA (Origin 9.0, Origin Lab Corp., USA) was carried out between the mean SOD (or CAT) proportions in the control and those in the chemical exposure from these independently repeated experiments. The probability levels of 0.05 were considered statistically significant (P < 0.05).

Notes

  1. When collecting the nematodes from nematode growth medium plates, the bacteria in the worm slurry would cause problems to the assays. Therefore, don’t use centrifugation, but use gravity settlement for sample collection. To fully get rid of the bacteria, gently wash the pellets (i.e., resuspend-settlement-discard supernatant) several times.
  2. Remember to avoid unnecessary stress on the nematodes. On one hand, use ice-cold PBS to lessen the stress on worms. On the other hand, it is better to spend less time on transfer and other performance with well-trained handling. Use a multi-channel pipette to make simultaneous addition of all solutions, so as to shorten the differences of reaction time among samples. Also, keep all the samples with the same performance to ensure the comparability between samples.
  3. Considering that the worms will stick to the tip and they tend to settle with gravity, resuspend the pellets with gentle and repetitive pipetting every time when transferring the worms. In this way, we can ensure that the worm density is uniform in the tube, and also that the worms stuck to the tip reach the equilibrium without further sticking.
  4. To ensure reproducibility and variability, there should be at least 100 nematodes in each tube.
  5. The supernatants from the homogenized PBS solutions of 100 nematodes are enough for four aliquots, which are adequate for four biochemical assays including the total protein. If more biochemical assays are performed simultaneously, the nematode numbers should increase.
  6. The protein concentration range of the protein standard solutions can be altered according to preliminary experiments, especially in cases where the nematode numbers are not counted.
  7. When homogenizing nematode samples, handle the pestles in a uniform manner among different samples. Keep the movement in the same strength with same time duration (10 sec homogenizing + 2 sec cooling down + 10 sec homogenizing for each tube), so as to improve the reproducibility of the data. Also, clean the pestle before using them for another tube, and keep the clean procedure uniform throughout the whole homogenizing procedure.
    Put pestles (for subsequent homogenization) in ice-cold PBS. After each use, wash the pestle in ice cold water, and put it back to the clean ice-cold PBS. This performance will ensure the cleanness of the pestle and also avoid potential influence of room-temperature pestles on protein stability of the samples.
  8. If the nematode number in each tube is much more than 500, the duration of homogenizing procedure should be extended to at least twice the normal time (e.g., 10 sec grinding + 2 sec cooling + 10 sec grinding + 2 sec cooling + 10 sec grinding + 2 sec cooling +10 sec grinding). The pestles should be washed with 400 μl PBS. Through this way, the homogenizing efficiency can be ensured, and the total protein concentration in the supernatants can be diluted to better perform the biochemical assays.
  9. In the prevention of water evaporation, a humid chamber will help.

Recipes

  1. Clorox solution
    Take 30 ml as an example. It consists of 0.6 g NaOH, 5 ml NaOCl (with 6-14% active Cl), and 25 ml deionized H2O, and the final concentrations are 0.5 M and ~1% for NaOH and NaOCl, respectively
  2. Phosphate buffered saline/buffer (PBS), pH 7.0
    If a ready-mix is not available, then make the solution as follows:
    1. Solution A: dissolve 17.418 g of K2HPO4 (FW = 174.18) in 1,000 ml in sterilized deionized water in sterilized bottle
    2. Solution B: dissolve 13.689 g of KH2PO4 (FW = 136.89) in 1,000 ml in sterilized deionized water in sterilized bottle
    3. In a 2,000 ml flask with a magnetic stir bar on a magnetic stir plate, add 750 ml solution B. After turning on the magnetic stirring apparatus, carefully put the probe of the pH meter underneath the water surface and above the magnetic stirrer. Then, add solution A into the flask slowly, and monitor the pH changes at the same time until the pH reaches 7.0
    4. If the pH overgoes 7.0, add more solution B slowly until the pH backs to 7.0

Acknowledgments

The authors are grateful for the financial supports by the National Natural Science Foundation of China (No. 21307095, No. 21407061), the International Science & Technology Cooperation Program of China (No. 2016YFE0123700), the Collaborative Innovation Center for Regional Environmental Quality, and the Swedish Research Council (contract Dnr. 639-2013-6913).

References

  1. Balaban, R. S., Nemoto, S. and Finkel, T. (2005). Mitochondria, oxidants, and aging. Cell 120(4): 483-495.
  2. Carretero, M., Solis, G.M., Petrascheck, M. (2017). C. elegans as model for drug discovery. Curr Top Med Chem 17: 1-10.
  3. Dengg, M. and van Meel, J. C. (2004). Caenorhabditis elegans as model system for rapid toxicity assessment of pharmaceutical compounds. J Pharmacol Toxicol Methods 50(3): 209-214.
  4. Feng, S., Cheng, H., Xu, Z., Shen, S., Yuan, M., Liu, J. and Ding, C. (2015). Thermal stress resistance and aging effects of Panax notoginseng polysaccharides on Caenorhabditis elegans. Int J Biol Macromol 81: 188-194.
  5. Solis, G. M. and Petrascheck, M. (2015). Measuring Caenorhabditis elegans life span in 96 well microtiter plates. J Vis Exp 49: e2496.
  6. Yu, Z., Chen, X., Zhang, J., Wang, R. and Yin, D. (2013a). Transgenerational effects of heavy metals on L3 larva of Caenorhabditis elegans with greater behavior and growth inhibitions in the progeny. Ecotoxicol Environ Saf 88: 178-184.
  7. Yu, Z., Sun, G., Liu, Y., Yin, D. and Zhang, J. (2017). Trans-generational influences of sulfamethoxazole on lifespan, reproduction and population growth of Caenorhabditis elegans. Ecotoxicol Environ Saf 135: 312-318.
  8. Yu, Z. Y., Zhang, J. and Yin, D. Q. (2012). Toxic and recovery effects of copper on Caenorhabditis elegans by various food-borne and water-borne pathways. Chemosphere 87(11): 1361-1367.
  9. Yu, Z., Zhang, J., Chen, X., Yin, D. and Deng, H. (2013b). Inhibitions on the behavior and growth of the nematode progeny after prenatal exposure to sulfonamides at micromolar concentrations. J Hazard Mater 250-251: 198-203.
  10. Yu, Z., Zhang, J. and Yin, D. (2016). Multigenerational effects of heavy metals on feeding, growth, initial reproduction and antioxidants in Caenorhabditis elegans. PLoS One 11(4): e0154529.

简介

超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性的测定被广泛用于表明测试化学品的毒性作用的抗氧化反应。 然而,早期的研究主要描述了根据制造商的说明进行的程序,而无需对任何生物体特异的修改。 本方案描述了分析线虫中超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性的步骤,它是可用于研究药物化合物和环境污染物的影响的模型生物。 主要步骤包括:(1)样品制备; (2)总蛋白测定; (3)SOD活性测定; (4)CAT活性测定; 和(5)中等名单和公式,以及数据分析和绩效说明。
【背景】生物标志物是对化学,药剂或治疗干预的响应而检查生物和病原过程至关重要的。生物体内的各种生物过程导致引起氧化应激的活性氧(ROS)。为了应对这种氧化应激,生物体可以部署超氧化物歧化酶(SOD)和过氧化氢酶(CAT)以清除ROS,以保护细胞的稳态(Balaban等,2005)。一方面,各种化学物质(污染物)可以阻止这种抗氧化反应,并扰乱包括人类在内的生物体的健康。另一方面,许多药物旨在加强抗氧化反应以改善健康。因此,SOD和CAT的活动对于反映化学品或/和药物的潜在影响非常重要。
   秀丽隐杆线虫(秀丽隐杆线虫)是一种用于研究药物化合物(Dengg和van Meel,2004; Carretero等,2017)和环境污染物(Yu et al。,2013a和2017)的影响的模型生物。几项研究使用SOD和CAT测定来表明抗氧化反应和潜在的机制,这些因素是测试化学物质的毒性作用(Feng et al。,2015; Yu et al。,2012; 2016和2017)。然而,这些研究简单地描述了通过使用通用试剂盒方案进行测定,而没有物种特异性修饰。因此,为了更好的具体指导,仍然需要在线虫中进行SOD和CAT测定的显式方案。
   在本协议中,我们提供了线虫协议和实验细节,以分析线虫中的SOD和CAT活性。

关键字:SOD, CAT, 总蛋白, 秀丽隐杆线虫, 实验方案

材料和试剂

  1. 移液器提示( https://online-shop.eppendorf.com )< br />
  2. 板(60毫米)
  3. 离心管,1.5 ml(Eppendorf,目录号:022364111)
  4. 吸收纸(KCWW,Kimberly-Clark,目录号:0131)
  5. 96孔板,盖子(Corning,Costar ®,目录号:3599)
  6. 密封胶带用于96孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:15036)
  7. 线虫(野生型N2)
    注意:这些线虫根据每个研究者的实验进行处理。在本协议中,线虫数量不同。
  8. BCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23225或23227)
    1. 蛋白质标准(5 mg / ml)
    2. BCA试剂A和B
  9. SOD测定试剂盒(Beyotime Biotechnology,目录号:S0101)
    1. 反应引发溶液
    2. SOD检测缓冲区
    3. WST-8
    4. 酶溶液
    5. 反应引发溶液(40x)
  10. CAT测定试剂盒(Beyotime Biotechnology,目录号:S0051)
    1. 酶结合物
    2. 洗液40x
    3. 基板A和B
    4. 停止解决方案
  11. 水合钠(NaOH),分析试剂(国药化学试剂,目录号:10019762)
  12. 次氯酸钠(Antiformin,NaOCl,6-14%活性Cl),分析试剂(ALADDIN,目录号:S101636-500 ml)
  13. 磷酸氢二钾(K 2 H 2 HPO 4),分析试剂(国药化学试剂,目录号:20032118)
  14. 磷酸二氢钾(KH 2 PO 4),分析试剂(国药化学试剂,目录号:10017618)
  15. Clorox溶液(参见食谱)
  16. 磷酸缓冲盐水/缓冲液(PBS),pH 7.0(参见食谱)

设备

  1. 移液器
  2. 显微镜
  3. 离心机,Eppendorf 5417R(Eppendorf,型号:5417 R,目录号:01396)
  4. Pestles,Eppendorf(Eppendorf,目录号:F0140010)
  5. 电机驱动式粉碎机(北京百湾电子科技有限公司,目录号:HG215-LH-A)
  6. 冰浴,离心管盒
  7. 酶标仪BioTek(BioTek Instruments,型号:Epoch)
  8. 灭菌瓶,Fisherbrand(100 ml,Fisher Scientific,目录号:FB800100; 250ml,Fisher Scientific,目录号:FB800250; 500ml,Fisher Scientific,目录号:FB800500)
  9. 磁力搅拌棒
  10. 磁力搅拌板
  11. pH计
  12. 孵化器,益恒(益恒,型号:LRH-1000F)

程序

  1. 样品制备
    1. 样本收集
      整个协议使用L4阶线虫作为参考点。根据早先的报告(Solis和Petrascheck,2015; Yu等人,2017),遵循线虫文化和年龄同步。从1.6毫升磷酸盐缓冲盐水(PBS,pH 7.0)的每个线虫生长培养基板(60毫米)上清洗L4阶段线虫到1.5毫升离心管中。在4℃下通过重力沉降30分钟后,小心地移出并丢弃上清液。加入500μl冰冷的PBS,用轻轻和重复的移液管再次悬浮沉淀,再沉淀15分钟。然后,丢弃上清液,加入500μl冰冷的PBS,用温和和重复的移液管重悬悬液,并在离心管中用200μlPBS中的20,50,100,200,300,400和500种线虫组分别使用以下步骤。
      以100条线虫为例。
      1. 在具有三次独立试验的显微镜下,在20μlPBS中计数线虫以估计整个管中的线虫密度。
      2. 根据获得的蠕虫数量,计算和调整PBS的体积,使最终密度为每20μl〜9〜11个蠕虫。例如,如果数字超过10,则相应添加更多的PBS;如果数量小于10,则执行10分钟的结算,并移除相应量的PBS以达到所需的密度。
      3. 将带有线虫的260μlPBS转移到带有移液管的新的1.5ml管中,并用轻轻和重复的移液管重新悬浮颗粒。
      4. 在三次独立试验中,在20μlPBS中计数线虫数,轻轻重复移液,确认线虫密度,在管中留下200μl。如果线虫密度与设置不一致,请重复步骤A1b。
      5. 通过相同的方式准备其他线虫编号的组。
    2. 样本存储
      将管以5,000×g离心5分钟(4℃)。用移液管小心丢弃上清液PBS,并将颗粒在-26℃过夜或-80℃保存一周以上。
    3. 样品均质
      1. 将干净的杵与马达驱动的组织研磨机连接起来。用冰冷的PBS清洗杵,并用吸收纸清洁。将杵紧紧地压在1.5ml离心管中的颗粒上。将离心管放在冰桶中(见图1)。启动组织研磨机,用2秒冷却,然后再进行10秒的研磨,研磨颗粒10秒。然后,使用200μl冰冷的PBS将杵上的残余液体回到离心管(见图1),然后将其从管中取出。
      2. 在4℃下将管以5,000xg离心5分钟。从每个样品中取出200μl上清液。将它们分成四个管,每个管有50μl,用于随后的测定。


        图1.样品均质。 左图显示电机驱动的组织研磨机在冰浴中使离心管中的颗粒均质化,右图显示残余液体用200μl冰冷的PBS洗脱杵。 >
  2. 总蛋白测定
    通过基于BCA方法的酶联免疫吸附测定(ELISA)试剂盒测量每个样品中的总蛋白(TP)。
    1. 蛋白质标准品的准备工作
      使用库存蛋白质标准品(5mg / ml)制备0.05,0.1,0.2,0.4,0.6,0.8,1.0,1.2mg / ml稀释液,每个使用PBS100μl。
    2. BCA工作解决方案的准备工作
      用BCA试剂A和B以50:1的体积比制备BCA工作溶液。 BCA工作溶液的总体积根据测试样品的量(每个样品200μl)计算。
    3. 蛋白质浓度的测定
      1. 对于每种蛋白质标准品,将20μl加入至少两次重复的96孔板中。使用20μlPBS缓冲液作为对照
      2. 对于每个线虫样品,从一个等分试样中加入20μl至少两次重复的96孔板。使用此等分试样以确保复制,并避免重复冻结样品的冻结。
      3. 然后,每孔加入200μlBCA工作溶液,所有蛋白质标准品和线虫样品。通过密封胶带密封孔以避免水分蒸发,并在37℃下将板孵育30分钟。
      4. 使用酶标仪测量562 nm处的吸光度(A <562 )
    4. 数据计算
      根据标准曲线计算样品中的蛋白质含量 线虫数与蛋白质浓度之间关系的一个介绍如图2所示

      图2.线虫数和线虫蛋白浓度之间的关系

  3. SOD活性分析
    通过基于WST-8方法的ELISA试剂盒测量每个样品中的SOD。 WST-8方法是基于WST-8的比色反应。在反应中,黄嘌呤氧化酶(XO)催化黄嘌呤的氧化转化,产生超氧阴离子,使WST-8猝灭,生成水溶性甲an(紫色染料)。由于SOD猝灭超氧化物阴离子,因此其活性抑制整体比色反应(见图3)。因此,抑制水平用于表示细胞,组织或其他生物样品中的SOD活性

    图3. WST-8与黄嘌呤的超氧阴离子的比色反应,SOD活性抑制比色反应

    1. 准备必要的解决方案
      根据制造商的ELISA试剂盒说明准备标准和控制措施以及工作解决方案。
      1. 每个样品需要160μlWST-8 /酶工作溶液和20μl反应引发溶液。根据线虫样本数量加上标准计算总量。对于WST-8 /酶工作溶液,160μl由151μlSOD检测缓冲液,8μlWST-8和1μl酶溶液组成。
      2. 通过将SOD检测缓冲液(即,即1μl原始溶液中的原始储备液1:40)稀释至39μl缓冲液来准备反应引发溶液。
    2. 样品测定
      1. 将20μl标准品和对照物添加至96孔板,至少重复两次。
      2. 将20μl线虫样品添加至96孔板,至少重复两次。值得注意的是,每个线虫样品在样品均质化步骤之后被处理并分成4个等分试样。使用一个等分试样以确保复制,并避免重复冻结样品的冻结。
      3. 使用多通道移液管在每个样品中加入160μlWST-8 /酶工作溶液,然后加入20μl反应引发工作溶液。标准品的浓度分别为100,50,10,5,2.5,2.25和0.625 U / ml
      4. 有两个空白控件。对于空白对照1,用SOD检测缓冲液代替20μl样品。对于空白对照2,用40μlSOD检测缓冲液代替20μl样品和20μl反应引发溶液
      5. 37℃孵育30分钟。
      6. 通过酶标仪读取450nm处的吸光度(A 450)
    3. SOD活性测定中的计算
      1. 抑制(%)=(空白对照1 标准或样品)/(空白对照1 空白对照2%)×100%。使用已知SOD活动的标准及其抑制来建立标准曲线。然后用曲线计算样品的SOD活性。标准曲线的一个例子如图4所示

        图4. WST-8比色反应的对数和抑制(%)SOD活性之间的关系

      2. 将每个线虫样品中的SOD酶活性作为同一样品的总蛋白(TP)的比例(P),以消除样品中线虫数的差异。线虫中标准化的SOD活性列在表1中。比例值一般在4.04〜4.35之间。具有20和50种线虫的组中的值比一般范围大得多。虽然这些值在平均值±3标准偏差(SD)之内,我们建议不要使用它们来避免不可靠性。这些结果表明,应使用至少100种线虫来确保测量SOD活动的可行性和稳定性。

        表1.标准化SOD活性与线虫号之间的连接


  4. CAT活性测定
    使用ELISA试剂盒测量每个样品中的CAT活性。测定原理是基于过氧化氢酶分解过氧化氢(H 2 O 2 O 2)的反应。过量的H 2 O 2 O 2可以与钼酸铵形成络合物以产生淡黄色溶液。溶液在450nm处的吸附可用于计算H 2 O 2 O 2的浓度,因此间接显示CAT活性。
    1. 试剂准备
      根据制造商的ELISA试剂盒说明,在测定前准备所有试剂,并根据样品数量,标准品和对照进行仔细计算。
    2. 添加标准品或样品
      将50μl标准品或样品加入96孔板中。使用50μlPBS作为空白对照。建议所有标准,样品和对照至少进行两次重复。值得注意的是,每个线虫样品在样品均质化步骤之后被处理并分成4个等分试样。使用一个等分试样以确保重复,并避免重复冻结样品。标准浓度分别为400,100,25,6.25,1.5625,0.4和0.1U / L
    3. 酶缀合
      在含有标准品,样品和空白的孔中加入100μl酶偶联物。然后通过密封胶带密封胶水,避免水分蒸发,并在37℃静置孵育微孔板60分钟。
      1. 丢弃液体内容物,然后用一张吸水纸将测定板敲打,以除去残留的缓冲液
      2. 加入350μl洗液(1x,用PBS从40x包装稀释),并保持微孔板静置1分钟。然后,丢弃液体内容物,然后将测试板在一张吸收纸上取出以除去残留的缓冲液
      3. 重复上述洗涤过程四次。
    4. 添加底物
      使用多通道移液管向每个孔中加入50μl底物A和50μl底物B。在37℃下在黑暗中孵育微孔板15分钟
    5. 端点测量
      向每个孔中加入50μl终止溶液。孵育1分钟后,在15分钟内用酶标仪测量450nm处的吸光度
    6. 数据计算和呈现
      根据标准曲线计算样品总CAT活性(见图5),其方法与SOD活性测定法相似。


      图5. H&lt;&gt;&lt; 2&gt; O 之间的比色反应的对数和抑制(CAT)之间的总CAT活性之间的关系> 2 钼酸铵

      将每个线虫样品中的CAT活性作为同一样品的总蛋白(TP)的比例(P),以消除样品中线虫数的差异。线虫中归一化的CAT活性列在表2中。比例值一般在8.74至10.36之间。具有20和50个线虫的组中的值显着大于该范围。这些结果表明,应使用至少100种线虫来确保测量CAT活动的可行性和稳定性
      表2.标准化CAT活动与线虫号之间的连接

数据分析

  1. 数据分析程序是指我们以前的报告(Yu等人,2013b)。首先,在至少三次重复测量每个样品的一个等分试样中的线虫总蛋白(TP)的值,并计算这些TP值(TP平均值)的平均值,以表示样品中的TP浓度。其次,同样样品的其他等分试样中的SOD(或CAT)值也至少在三次重复中测量。第三,将每个重复数据中的SOD(或CAT)值计算为其与TP平均值的比例,以获得SOD(或CAT)比例值。第四,计算SOD(或CAT)比例值以获得样品中的平均SOD(或CAT)比例和相应的标准误差。
  2. 在样品来自不同处理的情况下,来自对照的样品中的平均SOD(或CAT)比例归一化为100%(或1.0);然后,化学暴露样品中的SOD(或CAT)比例转化为对照(POC)的百分比(或折叠对照的变化);接下来,使用样本中的POC值(或折叠变化)来计算平均POC和相应的标准误差。
  3. 应从至少三次独立重复实验收集线虫样品,每个实验中都有一组数据。通过这些独立重复的实验,在对照组中的平均SOD(或CAT)比例和化学暴露量之间进行ANOVA(Origin 9.0,Origin Lab Corp.,USA)。 0.05的概率水平被认为具有统计学意义(P <0.05)

笔记

  1. 当从线虫生长培养基板收集线虫时,蠕虫浆液中的细菌会对测定产生问题。因此,不要使用离心,而是使用重力沉降来进行样品收集。为了完全摆脱细菌,轻轻洗涤沉淀(即重悬/沉淀 - 弃去上清液)几次。
  2. 记住要避免对线虫的不必要的压力。一方面,使用冰冷的PBS来减轻蠕虫的压力。另一方面,最好是花费更少的时间来转移和其他表演,训练有素的处理。使用多通道移液器同时添加所有溶液,以缩短样品间反应时间的差异。此外,保持所有样品具有相同的性能,以确保样品之间的可比性。
  3. 考虑到蠕虫会粘附在尖端,并且它们倾向于重力沉降,每当转移蠕虫时,都会以平缓和重复的移液来重悬。这样,我们可以确保管中的蠕虫密度是均匀的,并且粘附到尖端的蠕虫也能够在没有进一步粘附的情况下达到平衡。
  4. 为了确保重现性和变异性,每个管中应至少有100根线虫
  5. 来自100种线虫的均质化PBS溶液的上清液足够用于四个等分试样,其对于包括总蛋白质的四种生物化学测定是足够的。如果同时进行更多的生化测定,则线虫数量应该增加
  6. 蛋白质标准溶液的蛋白质浓度范围可以根据初步实验进行改变,特别是在不计算线虫数的情况下。
  7. 当均匀化线虫样品时,在不同样品中以均匀的方式处理杵。以相同的持续时间(10秒均化+ 2秒冷却+每个管均匀10秒)保持相同强度的运动,以提高数据的再现性。此外,在将其用于另一根管子之前清洁杵,并在整个均质程序中保持清洁程序均匀 将杵(用于随后均匀化)放入冰冷的PBS中。每次使用后,用冰冷的水清洗杵,并将其放回干净的冰冷PBS中。这种性能将确保杵的清洁度,并避免室温杵对样品蛋白质稳定性的潜在影响。
  8. 如果每个管中的线虫数大于500,则匀浆程序的持续时间应延长至正常时间的至少两倍(例如,10秒磨削+ 2秒冷却+ 10秒磨削+ 2秒冷却+ 10秒磨+ 2秒冷却+10秒磨)。杵应该用400μlPBS洗涤。通过这种方式,可以确保均质效率,并且可以稀释上清液中的总蛋白质浓度,以更好地进行生化测定。
  9. 在预防水分蒸发时,一个潮湿的房间将会有所帮助

食谱

  1. Clorox解决方案
    以30毫升为例。它由0.6g NaOH,5ml NaOCl(6-14%活性Cl)和25ml去离子H 2 O 2组成,最终浓度为0.5M,NaOH和NaOCl为〜1% ,分别是
  2. 磷酸盐缓冲盐水/缓冲液(PBS),pH 7.0
    如果没有可用的混合,那么请解决如下:
    1. 溶液A:在灭菌的无菌瓶中的无菌去离子水中,溶解17.418g K 2 N 2 HPO 4(FW = 174.18)
    2. 溶液B:在灭菌的无菌瓶中的灭菌去离子水中将13.689g KH 2 PO 4(FW = 136.89)溶解在无菌的去离子水中
    3. 在磁力搅拌板上带有磁力搅拌棒的2000毫升烧瓶中,加入750毫升溶液B.打开磁力搅拌装置后,小心地将pH计的探针放在水面下面和磁力搅拌器上方。然后缓慢加入溶液A,并同时监测pH变化,直到pH达到7.0
    4. 如果pH超过7.0,缓慢加入更多的溶液B,直到pH值回到7.0

致谢

作者非常感谢中国国家自然科学基金(21307095号,21407061),国际科学技术大学中国技术合作计划(No. 2016YFE0123700),区域环境质量合作创新中心和瑞典研究理事会(合同639-2013-6913)。

参考

  1. Balaban,RS,Nemoto,S.和Finkel,T。(2005)。线粒体,氧化剂和老化。 细胞 120(4):483-495。
  2. Carretero,M.,Solis,GM,Petrascheck,M。(2017)。 ℃。线虫作为药物发现的模型。 Curr Top Med Chem 17:1-10。
  3. Dengg,M.and van Meel,JC(2004)。 秀丽隐杆线虫作为药物化合物的快速毒性评估的模型系统。药物毒理学方法 50(3):209-214。
  4. Feng,S.,Cheng,H.,Xu,Z.,Shen,S.,Yuan,M.,Liu,J.and Ding,C.(2015)。 三七总皂甙多糖对秀丽隐杆线虫的耐热应力和老化作用< / em>。 Int J Biol Macromol 81:188-194。
  5. Solis,GM和Petrascheck,M。(2015)。测量96孔微量滴定板中的秀丽隐杆线虫的寿命。 J Vis Exp 49:e2496。
  6. Yu,Z.,Chen,X.,Zhang,J.,Wang,R. and Yin,D(2013a)。 重金属对秀丽隐杆线虫的L3幼虫的跨代影响,在后代中具有更大的行为和生长抑制作用。 > Ecotoxicol Environ Saf 88:178-184。
  7. Yu,Z.,Sun,G.,Liu,Y.,Yin,D. and Zhang,J.(2017)。 磺胺甲恶唑对秀丽隐杆线虫的寿命,繁殖和人口增长的跨代影响。生态毒素环境保护 135:312-318。
  8. Yu,ZY,Zhang,J. and Yin,DQ(2012)。各种食源性传播途径和水传播途径,铜对秀丽隐杆线虫的毒性和恢复作用。 87(11):1361- 1367.
  9. Yu,Z.,Zhang,J.,Chen,X.,Yin,D.and Deng,H。(2013b)。 抑制产后暴露于微摩尔浓度的磺酰胺的线虫后代的行为和生长危害物质 250 -251:198-203。
  10. Yu,Z.,Zhang,J.and Yin,D。(2016)。 重金属对秀丽隐杆线虫的饲养,生长,初始繁殖和抗氧化剂的多重影响。 11(4):e0154529 。
  • 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。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, J., Chen, R., Yu, Z. and Xue, L. (2017). Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans. Bio-protocol 7(16): e2505. DOI: 10.21769/BioProtoc.2505.
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。