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Peer-reviewed

Bronchoalveolar Lavage and Lung Tissue Digestion

支气管肺泡灌洗和肺组织消化

Hongwei Han Hongwei Han
SZ Steven F. Ziegler

发布: 2013年08月20日第3卷第16期 DOI: 10.21769/BioProtoc.859 浏览次数: 34777

评审: Anonymous reviewer(s)

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Abstract

Bronchoalveolar lavage (BAL) is a simple but valuable and typically performed technique commonly used for studying the pathogenesis of lung diseases such as asthma and COPD. Cell counts can be combined with new methods for examining inflammatory responses, such as ELISA, Flow cytometric analysis, immunohistochemistry, quantitative polymerase chain reaction, and HPLC to assess cellular expression for inflammatory cytokines and growth factor. Here we describe a basic procedure to collect BAL fluid and digest lung tissue for assessing a number of pulmonary components.

Keywords: Mouse (小鼠) search Lung function (肺功能) search Pulmonary disease (肺疾病) search Immune cell function (免疫细胞的功能) search Mucosal immunology (黏膜免疫) search

Materials and Reagents

  1. Mice
  2. Avertin (Sigma-Aldrich, catalog number: T48402 )
  3. PBS (Sigma-Aldrich, catalog number: D8537 )
  4. RPMI-1640 (Sigma-Aldrich, catalog number: R8758 )
  5. FACS buffer (PBS/0.5% BSA)
  6. Diff-Quick stain kit (Dade Behring, catalog number: B4132-1A )
  7. Single Cytology Funnels (Biomedical Polymers, Inc., catalog number: BMP-CYTO-S50 )
  8. Superfrost slides (Thermo Fisher Scientific, catalog number: 22-034-979 )
  9. Liberase Blendzyme (F. Hoffmann-La Roche, catalog number: 05401119001 )
  10. DNase (F. Hoffmann-La Roche, catalog number: 10104159001 )
  11. Trypan blue (Life Technologies, InvitrogenTM, catalog number: 15250-061 )
  12. Mouse Fc block (BD Biosciences, PharmingenTM, catalog number: 553141 )
  13. Antibodies for T cells (CD3+)
  14. Antibodies for B cells (B220+)
  15. Antibodies for eosinophils (Siglec-F+CD11c-)
  16. Antibodies for alveolar macrophages (AMs, Siglec-F+CD11c+CD11bintF4/80+)
  17. Antibodies for interstitial macrophages (Ims, Siglec-F-CD11c-CD11b+F4/80+)
  18. Antibodies for neutrophils (Siglec-F-CD11c-CD11b+Ly6G+)
  19. Antibodies for dendritic cells (DCs) (Siglec-F-CD11chiMHCIIhi)
  20. Erythrocyte lysis buffer (see Recipes)
  21. Tissue digestion solution (see Recipes)

Equipment

  1. 1 ml, 3 ml, and 10 ml sterile syringes
  2. 21 gauge lavage tube
  3. 21 gauge sterile needles
  4. Cotton thread No. 40
  5. 50 ml flask
  6. 15 ml and 50 ml conical tubes
  7. Microtubes
  8. 100 Micron cell strainer (BD Biosciences, Falcon®, catalog number: 352360 )
  9. Hemocytometer
  10. Centrifuge (Beckman Coulter, model: AllegraTM 6R )
  11. Cytospin cytocentrifuge (Thermo Fisher Scientific/Shandon, model: A7830002 )
  12. Microscope
  13. BD LSR II flow cytometer
  14. 3-Way Stopcocks (Bio-Rad Laboratories, model: 732-8103 )
  15. Magnetic stir bar (VWR International, model: 58949-006 )
  16. Multi-Position Magnetic Stirrers (VWR International, model: 12621-042 )

Procedure

English
中文翻译

文章信息

版权信息

© 2013 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Han, H. and Ziegler, S. F. (2013). Bronchoalveolar Lavage and Lung Tissue Digestion . Bio-protocol 3(16): e859. DOI: 10.21769/BioProtoc.859.

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分类

免疫学 > 免疫细胞分离

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