House Dust Mite Extract and Cytokine Instillation of Mouse Airways and Subsequent Cellular Analysis
对小鼠气道滴注尘螨提取物和细胞因子进行炎症分析
发布: 2016年07月20日第6卷第14期 DOI: 10.21769/BioProtoc.1875 浏览次数: 12858
评审: Ivan ZanoniMeenal SinhaMario M. Zaiss
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参见作者原研究论文
The authors used this protocol in:
Aug 2015
Abstract
Asthma is a complex disease of the airways primarily mediated by T helper 2 cells and innate lymphoid type 2 cells (Licona et al.,2013). Mice do not develop spontaneous asthma and therefore models have been developed for the assessment of key processes that underlie human pathology (Nial et al.,2008). Exposure to House Dust Mite (HDM) extract induces many key features of acute airway inflammation including elevated IgE levels, eosinophilia, goblet cell metaplasia, epithelial hypertrophy and airway hyperresponsiveness (AHR) in response to methacholine (Hammad et al., 2009; Dullaers et al., 2012; Coquet et al., 2015). The exact dose and duration of exposure to HDM can affect the type and extent of inflammation. In our case, we start with a low sensitizing dose that is increased on challenge, while others use differing schedules or a higher antigen concentration during sensitization of mice (Hondowicz et al., 2016; Trompette et al., 2014; Zaiss et al., 2015). We believe that using a low sensitizing dose more accurately separates the primary and secondary immune responses and reduces the possibility that HDM given during sensitization continues to fuel the immune response during challenge (Coquet et al., 2015; Plantinga et al., 2013). Here, we outline in text, pictures and video how to administer HDM extracts or cytokines via the intranasal route and briefly touch upon the subsequent analysis of inflammation in the airways [covered otherwise in ( Han et al., 2013)].
Keywords: HDM (HDM)
IL-33 (IL-33)
Asthma (哮喘)
Lavage (灌洗)
Airway Inflammation (气道炎症)
Materials and Reagents
- 1.5 ml Eppendorf tube (Sarstedt, catalog number: 72.269.001 )
- 100 μM Filter (BD, catalog number: 352360 )
Note: Currently, it is “Corning, FalconTM, catalog number: 352360”. - Sterile pipette tips (Corning, catalog number: 4804 )
- 23 Gauge (G) sterile needle (BD, catalog number: 300800 )
- Polyethylene tubing PE-10 (BD, IntramedicTM, catalog number: 427401 )
- 1 ml syringe (BD, catalog number: 309659 )
- 15 ml screw cap tube conical (Corning, FalconTM, catalog number: 352096 )
- 50 ml screw cap tube conical (Corning, FalconTM, catalog number: 352070 )
- Soft wood/styrofoam board
- Mice
Note: We typically utilize C57BL/6 mice of at least 4 weeks of age. Either sex can be used although it is best not to mix genders in a particular experiment. - Isoflurane (Baxter, catalog number: KDG9623 )
- House Dust Mite (HDM) extract (Greer Laboratories, catalog number: XPB70D3A2.5 )
- 1x Dulbecco’s phosphate buffered saline (DPBS) (Sigma-Aldrich, catalog number: D8537 )
- Ethanol
- Recombinant cytokines [e.g., rIL-21 (gifted by Zymogenetics); rIL-25 (Biolegend, catalog number: 587302 ) or IL-33 (Biolegend, catalog number: 580502 )]
- Fetal calf serum (FCS), heat inactivated (Sigma-Aldrich, catalog number: F4135 )
- Trypan blue (Sigma-Aldrich, catalog number: 93595 )
- Red blood cell lysis buffer (Sigma-Aldrich, catalog number: R7757 )
Equipment
- Laminar flow hood
- Rodent anesthesia system [e.g., Univentor 400 Anesthesia Unit or Univentor 410 Anesthesia unit (Univentor, catalog number: 8323101 )]
- Pipette p200 (Sigma-Aldrich, catalog number: Z678333 )
- Spray bottle with 70% ethanol (Sigma-Aldrich, catalog number: Z560847 )
- Surgical scissors (e.g., Sigma-Aldrich, catalog number: Z265969 )
- Surgical Thread 4-0 (0.17 mm), sterile (e.g., Agnthos, catalog number: 14757 )
- Stainless steel forceps (e.g., Sigma-Aldrich, catalog number: Z168785 )
- Hemocytometer
- Centrifuge [e.g., Hettich Rotina 420R (Sigma-Aldrich, catalog number: Z723630 )]
Figure 1. Anesthesia setup. A. Mice should be anesthetized by isoflurane with the rate of breathing carefully observed to ensure a sufficient degree of anesthesia without being too light or too deep. B. Example of anesthesia system in this case is the Univentor 400 with air flow set to 388 ml/min and Isoflurane concentration of 3.8%.
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Tibbitt, C. and Coquet, J. M. (2016). House Dust Mite Extract and Cytokine Instillation of Mouse Airways and Subsequent Cellular Analysis. Bio-protocol 6(14): e1875. DOI: 10.21769/BioProtoc.1875.
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分类
免疫学 > 动物模型 > 小鼠
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