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Superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1, is used to measure extracellular superoxide free radical release from various immune cells such as macrophage, neutrophils, microglia (brain macrophage) after stimulation. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

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[Bio101] Superoxide Measurement
[Bio101] 超氧化物的测定

神经科学 > 细胞机理
作者: Huiming Gao
Huiming GaoAffiliation: Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
11/20/2011, 5912 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.155

[Abstract] Superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1, is used to measure extracellular superoxide free radical release from various immune cells such as macrophage, neutrophils, microglia (brain macrophage) after stimulation. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

[Abstract] 超氧化物歧化酶(Superoxide dismutase,SOD)-对四氮唑盐WST-1可禁止的减少可用来测定诸如巨噬细胞,中性粒细胞,小胶质细胞(脑巨噬细胞)等各种免疫细胞受刺激后释放的细胞外超氧自由基。本实验方案经Dr. Hong实验室许多研究者多年发展和完善,特别是Dr. Bin Liu.

Materials and Reagents

  1. Phenol red-free HBSS (Life Technologies, InvitrogenTM, catalog number: 14175-095 )
  2. Superoxide dismutase from bovine erythrocytes (Sigma-Aldrich, catalog number: S7571-30KU )
  3. Phorbol 12-myristate 13-acetate (PMA)
  4. Lipopolysaccharide (LPS)
  5. Fresh WST-1 solution (see Recipes)
  6. SOD stock solution (see Recipes)

Equipment

  1. Microplate spectrophotometer (SpectraMax Plus)
  2. 96-well plates

Procedure

  1. Warm up the media or HBSS to 37 °C.
  2. Turn on the SpectraMax Plus microplate spectrophotometer, set the temperature to 37 °C.
  3. Set up the template for later reading according to your treatment paradigm (this step can also be done later during pretreatment of the cultures with drugs).
  4. Pretreat neuron-glia cultures, macrophages, or microglia grown in 96-well plates with desirable reagents in 100 μl phenol red-free treatment medium or HBSS for 30-60 min.
  5. Add PMA (a positive control), LPS (a common inflammogen), or vehicle in 50 μl of phenol red-free medium/HBSS to the cultures.
  6. Prepare fresh WST-1 solution in the darkroom, protecting the tube from light by covering it with foil.
  7. Add 50 μl of WST-1 (final concentration is 1 mM) in phenol red-free treatment medium/HBSS with or without 600 U ml-1 SOD to the cultures in the dark.
  8. The absorbance at 450 nm is read with a SpectraMax Plus microplate spectrophotometer.
    Note: Read multiple times to observe the dynamic changes of the absorbance. Suggested read time points: 0, 5, 10, 15, 20, 30, 45, 60, 90 min.

Recipes

  1. SOD stock solution
    Dissolve 30 KU of superoxide dismutase from bovine erythrocytes in 1 ml HBSS; store aliquots (100 μl) at -20 °C for later use.
  2. WST-1 solution (fresh, prepare right before use)

References

  1. Gao, H. M., Zhou, H., Zhang, F., Wilson, B. C., Kam, W. and Hong, J. S. (2011). HMGB1 acts on microglia Mac1 to mediate chronic neuroinflammation that drives progressive neurodegeneration. J Neurosci 31(3): 1081-1092.
  2. Zhang, F., Shi, J. S., Zhou, H., Wilson, B., Hong, J. S. and Gao, H. M. (2010). Resveratrol protects dopamine neurons against lipopolysaccharide-induced neurotoxicity through its anti-inflammatory actions. Mol Pharmacol 78(3): 466-477.

材料与试剂

 

1.        无酚红(Phenol red-free HBSS  (Invitrogen/Gibco 14175-095)

2.        牛红细胞超氧化物歧化酶(sigma, S7571-30KU)

 

设备

 

1.       微型分光光度计(SpectraMax Plus)

 

实验步骤

 

1.       加热培养基或HBSS  37oC

2.       打开SpectraMax Plus微孔板分光光度计, 设定温度为37oC

3.       根据处理规范为之后读数设定模板(这一步也可以在稍后的药物培养预处理过程中进行)

4.       预处理神经元的神经胶质细胞培养基,巨噬细胞,小胶质细胞生长于96孔板含有理想试剂的100 μl无酚红培养基或HBSS 30-60 min

5.        phorbol 12-myristate 13-acetate (PMA, 阳性对照),脂多糖 (LPS,一种常见的inflammogen),50 μl无酚红培养基或HBSS中的媒介物加入到培养基中。

6.       在暗室中准备新鲜的 WST-1溶液 ,将管外包上锡箔纸以避光保护。 

7.       加入50 μl  WST-1 (终浓度1 mM) 于无酚红 处理培养基或HBSS,黑暗中向培养基中加入或不加人 600 U/ml SOD 

8.       使用SpectraMax Plus微孔板分光光度计读取 450 nm 处的吸光度

注意:多次读取数据以观察吸光度动态变化。建议的读取数据时间点为: 0, 5, 10, 15, 20, 30, 45, 60, 90 min

 

配方

 

1.       SOD储液:溶解30 KU牛红细胞超氧化物歧化酶1 ml HBSS; 分成等份(100 μl)储存于-20oC供日后使用

2.       WST-1 溶液 (新鲜,使用前准备)

 

参考文献

 

1.        Zhang F., Shi J.S., Zhou H., Wilson B., Hong J.S., Gao H.M. (2010). Resveratrol protects dopamine neurons against lipopolysaccharide-induced neurotoxicity through its anti-inflammatory actions. Molecular Pharmacology78(3): 466-77. 

2.        Gao H.M., Zhou H., Zhang F., Wilson B.C., Kam W., Hong J.S. (2011). HMGB1 acts on microglia Mac1 to mediate chronic neuroinflammation that drives progressive neurodegeneration. Journal of Neuroscience 31(3): 1081-92. 

 

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How to cite this protocol: Gao, H. (2011). Superoxide Measurement. Bio-protocol Bio101: e155. DOI: 10.21769/BioProtoc.155; Full Text



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