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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.

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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

免疫学 > 动物模型 > 小鼠
作者: Zheng Liu
Zheng LiuAffiliation: The Feinstein Institute for Medical Research, Manhasset, NY, USA
For correspondence: zl2119@caa.columbia.edu
Bio-protocol author page: a12
Anne Davidson Lab, 6/20/2011, 17352 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.84

[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)

[Abstract] 腹膜内注射巯基乙酸盐可以稳定诱导大量的中性白细胞到腹腔中。对这些细胞的运输被认为是通过CXCL1 ,CXCL2, 和 CXCL8等炎症趋化因子 介导的(1, 2)。 因此这一模型可以用于在生物工程小鼠(例如敲除或转基因小鼠)中检测中性白细胞向这些炎症趋化因子移动,或者能够用于检测确定的分子(例如一些小分子或者抑制类抗体)抑制这些因子的化学活性. 这一实验方法被作者成功的用于鉴定病毒的多重的趋化因子抑制剂的功能。

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.

材料和试剂

 

I. 抗体

1.        Rat anti-mouse Gr-1 PE (BD Biosciences 553129)

2.        Rat anti-mouse CD11b FITC (Southern Biotech 1560-02)

II. 其它材料

3.        消毒的4% 溶于ddH2O的巯基乙酸盐(Sigma 70157)

注意: 巯基乙酸盐溶液需要用锡箔纸包好后避光置于室温下保持,可以保存几个星期直到变成棕色。变质可以影响到对腹膜炎的诱导作用。

 

仪器

 

1.        BD LSR II 流式细胞测定器

 

步骤

 

1.        向小鼠腹膜内注射1ml消毒了的4%的巯基乙酸盐。

2.        两个小时后,麻醉小鼠.

中性粒白细胞将在这个时间点左右达到高峰。实验者如果想观察到单核细胞的流入需要在麻醉小鼠前等48小时。

3.        在小鼠的下腹部切开个小口露出肌肉。

注意: 不要危害的腹腔的完整性。

4.        26G1/2 针慢慢的向腹腔注射10ml冰上预冷的PBS

注意: 一些实验方案建议PBS中包含低浓度的EDTA去激活腹膜细胞的最大程度的产出量.

5.        拔掉注射针.

6.        通过抓住尾巴来固定小鼠并搅拌液体3分钟尽大范围的清洗小鼠的腹腔。

7.        将小鼠放到一旁并插入一个带有 18G1/2针头的10ml 注射器.

8.        慢慢的拔动注射器尽可能多的回收PBS

9.        记录回收的PBS体积.

10.    1细胞5min

11.    弃掉上清并用 100 ul  含有1:200 anti-GR-1 PE  anti-CD11b FITC抗体的3% FBS 重悬。

12.    在室温下孵育15 min

13.    1ml PBS洗涤 并且2004oC 离心5min

14.    弃掉上清用 200ul PBS重悬细胞。

15.    在流式细胞仪上得到这完整的200ul细胞量。

16.    计算 Gr-1highCD11bhigh 细胞数目并且根据回收到PBS体积来调整数量。

例如如果回收的PBS体积为9ml那么腹腔中全部的中性粒白细胞的数目=(计算的Gr-1highCD11bhigh细胞数x 10ml)/9ml

注意: 一些实验方案简单的通过 用血球计数器来数细胞。 但这一结果可能不能准确的反映腹腔中中性粒细胞的数目,原因是腹腔中还有其它种类的细胞如B1 B细胞和巨噬细胞。

 

参考文献

 

1.        Call D.R., Nemzek J.A., Ebong S.J., Bolgos G.L., Newcomb D.E., Remick D.G. (2001). Ratio of local to systemic chemokine concentrations regulates neutrophil recruitment. American Journal of Pathology 158(2): 715-21. 

2.        Cacalano G., Lee J., Kikly K., Ryan A.M., Pitts-Meek S., Hultgren B., Wood W.I., Moore M.W. (1994). Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog. Science 265(5172): 682-4. 

 

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How to cite this protocol: Liu, Z. (2011). Thioglycollate Induced Peritonitis. Bio-protocol Bio101: e84. DOI: 10.21769/BioProtoc.84; Full Text



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8/29/2016 7:58:31 AM  

achille Broggi
Boston Children's Hospital HMS

How many neutrophils are recovered per mouse?

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7/11/2012 3:22:45 AM  

@ guest: nobody knows.

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5/18/2012 5:08:46 AM  

why thioglycollate elicits a robust influx of neutrophils into peritoneal cavity? what's the mechanism?

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