欢迎您, 登录 | 注册

首页 | English

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[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, 6525 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(SOD) - 抑制性还原的四唑盐,WST-1,用于测量刺激后从各种免疫细胞如巨噬细胞,中性粒细胞,小胶质细胞(脑巨噬细胞)的细胞外超氧自由基释放。 这个协议已经由Hong博士实验室的各种研究人员,尤其是刘博士多年来开发和改进。

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.

材料和试剂

  1. 无酚红HBSS(Life Technologies,Invitrogen TM ,目录号:14175-095)
  2. 来自牛红细胞的超氧化物歧化酶(Sigma-Aldrich,目录号:S7571-30KU)
  3. 佛波醇12-豆蔻酸酯13-乙酸酯(PMA)
  4. 脂多糖(LPS)
  5. 新鲜的WST-1解决方案(参见配方)
  6. SOD储备溶液(见配方)

设备

  1. 微孔板分光光度计(SpectraMax Plus)
  2. 96孔板

程序

  1. 将介质或HBSS预热至37°C
  2. 打开SpectraMax Plus微孔板分光光度计,将温度设置为37°C
  3. 根据您的治疗范例设置模板以供稍后阅读(此步骤也可以在药物预处理过程中稍后进行)。
  4. 用100μl无酚红处理培养基或HBSS中的所需试剂在96孔板中预先培养神经元胶质细胞培养物,巨噬细胞或小胶质细胞30-60分钟。
  5. 在培养物中加入50μl无酚红培养基/HBSS中的PMA(阳性对照),LPS(常见的致炎物质)或载体。
  6. 在暗室中制备新鲜的WST-1溶液,通过用箔覆盖保护管免受光。
  7. 在黑暗中向培养物中加入50μl不含酚红的处理培养基/HBSS中的50μlWST-1(终浓度为1mM),有或没有600U/ml的SOD。
  8. 用SpectraMax Plus微孔板分光光度计读取450nm处的吸光度 注意:多次读取以观察吸光度的动态变化。 建议读取时间点:0,5,10,15,20,30,45,60,90分钟。

食谱

  1. SOD储液
    在1ml HBSS中溶解30KU来自牛红细胞的超氧化物歧化酶; 将等分试样(100μl)储存在-20℃备用
  2. WST-1溶液(新鲜,使用前准备好)

参考文献

  1. Gao,H. M.,Zhou,H.,Zhang,F.,Wilson,B.C.,Kam,W.and Hong,J.S。(2011)。 HMGB1作用于小胶质细胞Mac1,以调节慢性神经炎症,从而驱动进行性神经变性。 J Neurosci 31(3):1081-1092。
  2. Zhang,F.,Shi,J.S.,Zhou,H.,Wilson,B.,Hong,J.S。和Gao,H.M。 白藜芦醇通过其抗炎作用保护多巴胺神经元免受脂多糖诱导的神经毒性。 em> Mol Pharmacol 78(3):466-477。
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。

X


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



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)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。


登陆 | 注册