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[Bio101] Thioglycollate Induced Peritonitis
[Bio101] 利用巯基乙酸盐诱导建立腹膜炎模型   

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Abstract

Intraperitoneal (i.p.) injection of thioglycollate elicits a robust influx of neutrophils into peritoneal cavity. The trafficking of the cells is believed to be mediated by chemokines CXCL1, CXCL2, and CXCL8 (Call et al., 2001; Cacalano et al., 1994). Thus this model can be used to test the ability of neutrophils to migrate towards these chemokines in bioengineered mouse strains (e.g. knockout or transgenic mice) or the ability of certain molecules to inhibit the chemoattractive activities of these chemokines (e.g. small molecules or inhibitory antibodies). This protocol has been used by the author successfully to test the functions of a viral multi-chemokine inhibitor.

Keywords: Mouse model(小鼠模型), Peritonitis(腹膜炎), Inflammation(炎症), Thioglycollate(巯基乙酸)

Materials and Reagents

  1. Antibodies
    1. Rat anti-mouse Gr-1 PE (BD Biosciences, catalog number: 553129 )
    2. Rat anti-mouse CD11b FITC (Southern Biotech, catalog number: 1560-02 )

  2. Other materials
    1. Mice
    2. PBS
    3. 4% sterile thioglycollate (Sigma-Aldrich, catalog number: 70157 ) in ddH2O
      Note: Thioglycollate solution needs to be wrapped with aluminum foil to avoid light and be placed at room temperature to age for several weeks until it turns to brown in color. The aging process is critical to the ability of thioglycollate to induce peritonitis.

Equipment

  1. 6G1/2 needle
  2. 18G1/2 needle connected with a 10 ml syringe
  3. BD LSR II flow cytometer

Procedure

  1. Inject intraperitoneally mice with 1 ml of 4% sterile thioglycollate.
  2. Two hour later, anesthetize the mice.
    The influx of neutrophils is at peak around this time point. Users need to wait for 48 h before they anesthetize the mice, should they wish to observe monocyte influx.
  3. Cut a small opening at the lower abdomen to expose the underneath muscle.
    Note: Do not compromise the integrity of peritoneal cavity.
  4. Slowly inject 10 ml ice cold PBS into peritoneal cavity using a 26G1/2 needle.
    Note: Some protocols suggest using PBS containing low concentrations of EDTA to achieve maximal yield of peritoneal cells.
  5. Remove the needle.
  6. Hold the mouse by tail and swish around for 3 min to wash peritoneal cavity extensively.
  7. Lay the mouse by the side and insert an 18G1/2 needle connected with a 10 ml syringe.
  8. Retrieve maximal amount of PBS by slowly pulling out the plunge.
  9. Record the volume of PBS retrieved.
  10. Spin down the cells at 1,200 RMP at 4 °C for 5 min.
  11. Discard supernatant and resuspend cells with 100 μl of 3% FBS containing 1: 200 anti-GR-1 PE and anti-CD11b FITC.
  12. Incubate at room temperature for 15 min.
  13. Wash with 1 ml PBS and spin down at 1,200 RMP at 4 °C for 5 min.
  14. Discard supernatant and resuspend cells with 200 μl PBS.
  15. Acquire the entire 200 μl of cells on flow cytometer.
  16. Calculate the numbers of Gr-1highCD11bhigh cells and adjust the numbers to the volume of retrieved PBS.
    I.e. if the volume of retrieved PBS is 9 ml, then the total number of neutrophils per peritoneal cavity = (calculated number of Gr-1highCD11bhigh cells x 10 ml)/9 ml

Notes

Some protocols prefer to decide cell numbers simple by counting cells using hemocytometer. The numbers obtained by such method may not accurately reflect the number of neutrophils because there are other types of cells such as B1 B cells and macrophages in peritoneal cavity.

Acknowledgments

This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).

References

  1. Call, D. R., Nemzek, J. A., Ebong, S. J., Bolgos, G. L., Newcomb, D. E. and Remick, D. G. (2001). Ratio of local to systemic chemokine concentrations regulates neutrophil recruitment. Am J Pathol 158(2): 715-721.
  2. Cacalano, G., Lee, J., Kikly, K., Ryan, A. M., Pitts-Meek, S., Hultgren, B., Wood, W. I. and Moore, M. W. (1994). Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog. Science 265(5172): 682-684.

简介

腹膜内(i.p.)注射巯基乙酸盐引发嗜中性粒细胞进入腹膜腔的强烈内流。 认为细胞的运输由趋化因子CXCL1,CXCL2和CXCL8介导(Call等人,2001; Cacalano等人,1994)。 因此,该模型可用于测试中性粒细胞在生物工程化小鼠品系(例如敲除或转基因小鼠)中向这些趋化因子迁移的能力或某些分子抑制这些趋化因子的化学吸引活性的能力(例如, 例如小分子或抑制性抗体)。 该方案已被作者成功地用于测试病毒多趋化因子抑制剂的功能。

关键字:小鼠模型, 腹膜炎, 炎症, 巯基乙酸

材料和试剂

  1. 抗体
    1. 大鼠抗小鼠Gr-1PE(BD Biosciences,目录号:553129)
    2. 大鼠抗小鼠CD11b FITC(Southern Biotech,目录号:1560-02)

  2. 其他材料
    1. 小鼠
    2. PBS
    3. 在ddH 2 O中的4%无菌巯基乙酸盐(Sigma-Aldrich,目录号:70157) 注意:硫代乙醇酸盐溶液需要用铝箔包裹以避免光照,并放置在室温下老化几个星期,直到变成棕色。 老化过程对于巯基乙酸盐诱导腹膜炎的能力是至关重要的。

设备

  1. 6G1/2针
  2. 18G1/2针用10ml注射器连接
  3. BD LSR II流式细胞仪

程序

  1. 用1ml 4%无菌巯基乙酸盐注射腹腔内小鼠
  2. 两小时后,麻醉小鼠。
    嗜中性粒细胞的流入在该时间点附近处于峰值。 用户需要等待48小时,他们麻醉小鼠,如果他们希望观察单核细胞流入。
  3. 在下腹部切开一个小开口,暴露下面的肌肉。
    注意:不要损害腹膜腔的完整性。
  4. 使用26G1/2针缓慢注射10ml冰冷PBS到腹腔 注意:一些方案建议使用含有低浓度EDTA的PBS以达到腹膜细胞的最大产量。
  5. 取下针。
  6. 用尾巴握住鼠标,大约3分钟,彻底清洗腹膜腔
  7. 放置鼠标侧面,并插入用10毫升注射器连接的18G1/2针。
  8. 通过缓慢地拔出浸出物来收回最大量的PBS。
  9. 记录检索的PBS的体积。
  10. 在4℃下在1,200RMP下将细胞旋转5分钟
  11. 弃去上清液并用100μl含有1:200抗GR-1PE和抗CD11b FITC的3%FBS重悬细胞。
  12. 在室温下孵育15分钟
  13. 用1ml PBS洗涤并在1,200RMP在4℃下旋转5分钟
  14. 弃去上清液并用200μlPBS重悬细胞
  15. 在流式细胞仪上获得整个200微升的细胞
  16. 计算Gr-1 CD11b 细胞的数量,并将数量调整为检索到的PBS的体积。
    如果检索的PBS的体积是9ml,则每个腹膜腔的嗜中性粒细胞的总数=(计算出的Gr-1高CD11b高 sup>细胞×10ml)/9ml

笔记

一些协议更喜欢通过使用血细胞计数器计数细胞来简单地决定细胞数量。 通过这种方法获得的数目可能不准确地反映嗜中性粒细胞的数目,因为在腹膜腔中存在其它类型的细胞,例如B1B细胞和巨噬细胞。

致谢

该方案在Anne Davidson博士在Feinstein Institute for Medical Research,NY,USA的实验室中开发或修改。这项工作得到NY SLE基金会(RB),Rheuminations,NIH AI082037和AR 049938-01,NIH(PO1 AI51392和PO1 AI51392的流式细胞术和蛋白质表达和四聚体核)的赠予的支持。

参考文献

  1. Call,D.R.,Nemzek,J.A.,Ebong,S.J.,Bolgos,G.L.,Newcomb,D.E。和Remick,D.G。(2001)。 局部与系统性趋化因子浓度比例调节嗜中性粒细胞募集。 Am J Pathol 158(2):715-721。
  2. Cacalano,G.,Lee,J.,Kikly,K.,Ryan,A.M.,Pitts-Meek,S.,Hultgren,B.,Wood,W.I.and Moore,M.W。(1994)。 中性粒细胞和B细胞在缺乏鼠IL-8受体同源基因的小鼠中的扩增。 科学 265(5172):682-684
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, Z. (2011). Thioglycollate Induced Peritonitis. Bio-protocol Bio101: e84. DOI: 10.21769/BioProtoc.84;
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Achille Broggi
Boston Children’s Hospital
How many neutrophils are recovered per mouse?
8/29/2016 7:58:31 AM Reply
@ guest: nobody knows.
7/11/2012 3:22:45 AM Reply
why thioglycollate elicits a robust influx of neutrophils into peritoneal cavity? what's the mechanism?
5/18/2012 5:08:46 AM Reply