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Pulmonary Myeloperoxidase Activity
肺髓过氧化物酶活性试验   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Oct 2012

Abstract

Neutrophils are considered one of the first responders of the innate immune response. Their primary activities are to migrate to sites of infection by chemotaxis and trans-migration across the endothelium (Gaines et al., 2005). Once at the site of infection, they phagocytize microbes and kill them. Critical to the neutrophil’s ability to kill microbes are the multiple degradative enzymes contained within granules. The activity of these enzymes is non-specific, and therefore, neutrophils also contribute to tissue damage at the site of infection (Gaines and Berliner, 2005). Measurement of neutrophil infiltration into tissues is one way to gauge the severity of infection, inflammation, and tissue damage (Ayala et al., 2002). Myeloperoxidase is found in the primary granules of neutrophils and is an effective measure of neutrophil infiltration into tissues (Gaines and Berliner, 2005).

Keywords: Neutrophil (中性粒细胞), Inflammation (炎症), Lung injury (肺损伤)

Materials and Reagents

  1. Fresh or snap-frozen tissues
  2. Liquid nitrogen
  3. MPO Fluoremetric Detection Kit (Assay Designs, catalog number: 907-029 )
  4. N-ethylmaleimide (Sigma-Aldrich, catalog number: E2068 )
  5. Hexadecyltrimethylammonium (Sigma-Aldrich, catalog number: 52366 )
  6. Deionized water
  7. DMSO (Sigma-Aldrich, catalog number: D8418 )
  8. Assay buffer (see Recipes)
  9. Hydrogen Peroxide (see Recipes)
  10. Detection Reagent (see Recipes)
  11. Reaction cocktail (see Recipes)

Equipment

  1. 14 ml Polystyrene test tubes
  2. 2 ml Microfuge tubes
  3. Black-welled 96 well plates
  4. Polytron homogenizer
  5. Sonicator
  6. Fluorescent plate reader
  7. Refrigerated centrifuge

Procedure

  1. Weigh out 50 mg tissue into polycarbonate test tubes containing 0.5-1 ml ice cold 1x assay buffer with 10 mM N-ethylmaleimide.
  2. Homogenize to just disrupt the tissue by placing the tip of the homogenizer in the bottom of the tube and switching the machine on, agitating the tube to move the tip of the homogenizer throughout the liquid and then switching the machine off after approximately one sec. Repeat until the tissues are just disrupted, usually 9 more times for a total of 10 pulses.
  3. Centrifuge at 500 x g for 10 min at 4 °C.
  4. Discard supernatant, add 500 μl ice cold 1x assay buffer containing 0.5% hexadecyltrimethylammonium to the pellet and transfer to a 2 ml microfuge tube.
  5. Homogenize to lyse the cells by placing the tip of the homogenizer in the bottom of the tube and switching the machine on, agitating the tube to move the tip of the homogenizer throughout the liquid and then switching the machine off after approximately 5 sec. Repeat 9 times for a total of 10 pulses. Place on ice.
  6. Sonicate to further lyse the cells at 50% power for 10 sec. Place on ice. Repeat 2 times for a total of 3 pulses.  
  7. Snap freeze in liquid nitrogen and thaw at room temperature by placing in liquid nitrogen, immediately removing from the nitrogen and leave the samples at room temperature until completely thawed.  Repeat snap freeze and thaw once.
  8. Store at -80 °C until assayed.
  9. Prior to assay, serially dilute the included MPO standard in assay buffer.
  10. To assay, add 50 μl of sample or the MPO standard to the bottom of a black 96 well plate. Add 50 μl Reaction Cocktail provided by the kit. All samples and standards should be run in duplicate.
  11. Incubate at room temperature in the dark for 30 min.  
  12. Read the fluorescence at 550 nm excitation and 595 nm emission every 10 min until 60 min incubation.
  13. Plot the standard concentration vs. the maximum relative fluorescence units to create a standard curve (Figure 1).

     
    Figure 1. Representative Standard Curve

  14. Determine the concentration of the unknowns from the standard curve.

Recipes

  1. 1x Assay buffer   
    4 ml MPO assay buffer concentrate
    36 ml deionized water
  2. Hydrogen Peroxide
    22.7 μl 3% hydrogen peroxide
    977 μl deionized water
  3. Detection Reagent
    Supplied vial
    500 μl DMSO
  4. Reaction cocktail
    50 μl detection reagent
    5 μl hydrogen peroxide
    4.875 ml 1x assay buffer

Acknowledgments

This protocol is adapted from Ozment et al. (2012).

References

  1. Ayala, A., Chung, C. S., Lomas, J. L., Song, G. Y., Doughty, L. A., Gregory, S. H., Cioffi, W. G., LeBlanc, B. W., Reichner, J., Simms, H. H. and Grutkoski, P. S. (2002). Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency. Am J Pathol 161(6): 2283-2294.    
  2. Gaines, P., Chi, J. and Berliner, N. (2005). Heterogeneity of functional responses in differentiated myeloid cell lines reveals EPRO cells as a valid model of murine neutrophil functional activation. J Leukoc Biol 77(5): 669-679.
  3. Ozment, T. R., Ha, T., Breuel, K. F., Ford, T. R., Ferguson, D. A., Kalbfleisch, J., Schweitzer, J. B., Kelley, J. L., Li, C. and Williams, D. L. (2012). Scavenger receptor class a plays a central role in mediating mortality and the development of the pro-inflammatory phenotype in polymicrobial sepsis. PLoS Pathog 8(10): e1002967.    

简介

嗜中性粒细胞被认为是先天免疫反应的第一反应者之一。 它们的主要活性是通过趋化性和穿过内皮的转移迁移到感染部位(Gaines等人,2005)。 一旦在感染的部位,他们吞噬微生物并杀死他们。 中性粒细胞杀死微生物的能力的关键是颗粒内包含的多种降解酶。 这些酶的活性是非特异性的,因此,嗜中性粒细胞也有助于感染部位的组织损伤(Gaines和Berliner,2005)。 中性粒细胞浸润到组织中的测量是测量感染,炎症和组织损伤的严重性的一种方式(Ayala等人,2002)。 髓过氧化物酶存在于嗜中性粒细胞的原始颗粒中,并且是嗜中性粒细胞浸润到组织中的有效量度(Gaines和Berliner,2005)。

关键字:中性粒细胞, 炎症, 肺损伤

材料和试剂

  1. 新鲜或速冻组织
  2. 液氮
  3. MPO荧光检测试剂盒(Assay Designs,目录号:907-029)
  4. N-乙基马来酰亚胺(Sigma-Aldrich,目录号:E2068)
  5. 十六烷基三甲基铵(Sigma-Aldrich,目录号:52366)
  6. 去离子水
  7. DMSO(Sigma-Aldrich,目录号:D8418)
  8. 测试缓冲区(参见配方)
  9. 过氧化氢(参见配方)
  10. 检测试剂(参见配方)
  11. 反应鸡尾酒(见配方)

设备

  1. 14 ml聚苯乙烯试管
  2. 2 ml Microfuge管
  3. 黑色96孔板
  4. Polytron匀浆器
  5. 超声波仪
  6. 荧光平板读取器
  7. 冷冻离心机

程序

  1. 将50mg组织称重到含有0.5-1ml冰冷的含10mM N-乙基马来酰亚胺的测定缓冲液的聚碳酸酯试管中。
  2. 通过将均化器的尖端放置在管的底部并打开机器,搅拌管以将均化器的尖端移动穿过液体,然后在约1秒后关闭机器,从而均匀化以破坏组织。 重复直到组织被破坏,通常9次,总共10个脉冲
  3. 在4℃下以500×g离心10分钟。
  4. 弃去上清液,加入500μl冰冷的含有0.5%十六烷基三甲基铵的1x测定缓冲液到沉淀中,并转移到2ml微量离心管中。
  5. 通过将均化器的尖端放置在管的底部并切换机器,搅拌管以将均化器的尖端移过整个液体,然后在约5秒后关闭机器,从而均质化以裂解细胞。重复9次,共10个脉冲。放在冰上。
  6. 超声以进一步裂解细胞在50%功率10秒。放置在冰上。重复2次,共3个脉冲。  
  7. 在液氮中快速冷冻并在室温下通过置于液氮中解冻,立即从氮气中除去并将样品在室温下放置直至完全解冻。重复快照冻结和解冻一次。
  8. 储存于-80℃直至测定。
  9. 在测定之前,在测定缓冲液中连续稀释所包含的MPO标准品
  10. 为了测定,将50μl样品或MPO标准品加入黑色96孔板的底部。 加入试剂盒提供的50μl反应混合物。 所有样品和标准品应重复运行。
  11. 在室温下在黑暗中孵育30分钟。  
  12. 读取荧光在550 nm激发和595 nm发射每10分钟,直到60分钟孵育
  13. 绘制标准浓度与最大相对荧光单位,以创建标准曲线(图1)。

     
    图1.代表性标准曲线

  14. 从标准曲线确定未知物的浓度。

食谱

  1. 1x测试缓冲区  
    4ml MPO测定缓冲液浓缩物
    36ml去离子水
  2. 过氧化氢
    22.7μl3%过氧化氢
    977μl去离子水
  3. 检测试剂
    供应小瓶
    500μlDMSO
  4. 反应液
    50μl检测试剂
    5μl过氧化氢
    4.875ml 1x测定缓冲液

致谢

该协议改编自Ozment等人(2012)。

参考文献

  1. Ayala,A.,Chung,C.S.,Lomas,J.L.,Song,G.Y.,Doughty,L.A.,Gregory,S.H.,Cioffi,W.G.,LeBlanc,B.W.,Reichner,J.,Simms,H.H.and Grutkoski,P.S.(2002)。 Shock诱导的嗜中性粒细胞介导的对小鼠急性肺损伤的初免:TLR-4和TLR的发散效应-4/FasL缺陷。 m Pathol 161(6):2283-2294。    
  2. Gaines,P.,Chi,J.and Berliner,N。(2005)。 分化骨髓细胞系中功能反应的异质性揭示EPRO细胞作为鼠中性粒细胞功能活化的有效模型。 Leukoc Biol 77(5):669-679。
  3. Ozment,TR,Ha,T.,Breuel,KF,Ford,TR,Ferguson,DA,Kalbfleisch,J.,Schweitzer,JB,Kelley,JL,Li,C.and Williams,DL(2012) "_blank"href ="http://www.ncbi.nlm.nih.gov/pubmed/23071440">清道夫受体类a在介导多种微生物败血症的死亡率和促炎表型的发展中起中心作用a>。 PLoS Pathog 8(10):e1002967。    
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Copyright: © 2013 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. Ozment, T. R. (2013). Pulmonary Myeloperoxidase Activity. Bio-protocol 3(15): e851. DOI: 10.21769/BioProtoc.851.
  2. Ozment, T. R., Ha, T., Breuel, K. F., Ford, T. R., Ferguson, D. A., Kalbfleisch, J., Schweitzer, J. B., Kelley, J. L., Li, C. and Williams, D. L. (2012). Scavenger receptor class a plays a central role in mediating mortality and the development of the pro-inflammatory phenotype in polymicrobial sepsis. PLoS Pathog 8(10): e1002967.
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