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Bronchoalveolar Lavage and Lung Tissue Digestion
支气管肺泡灌洗和肺组织消化   

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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(鼠标), Lung function(肺功能), Pulmonary disease(肺疾病), Immune cell function(免疫细胞的功能), Mucosal immunology(黏膜免疫)

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

  1. Anesthetize the mouse by intraperitoneal injection of 1 ml 2.5% Avertin in PBS.
  2. Using scissors to expose thoracic cage and neck. Dissect tissue from neck to expose trachea.
  3. Proceed to open the diaphragm by cutting the rib cage to expose both the heart and lungs. Take caution not to pierce heart or lungs.
  4. Use forceps to slide 1 inch long piece of thread underneath trachea.
  5. Make a small incision in the trachea, to allow passage of 21 gauge lavage tube into trachea. The distance between the proximal end of the trachea and the tracheal incision is 2-3 mm.
    Note: Do not cut trachea all the way through.
  6. Cut a 1-1.5 inch segment of 21 gauge tube, carefully pass a 21 gauge needle into the tubing. Insert tubing into trachea and tie thread into single knot around tubing in trachea.
  7. Slowly inject 1 ml cold PBS with 0.1 mM EDTA into lungs using “input” 3 ml syringe via 3-way stopcocks: Watch lungs inflating and do not overinflate.
  8. Collect ~1 ml BAL fluid (BALF) from lungs using “output” 3 ml syringe into microtubes on ice.
  9. Repeat steps 7-8 for 3 washes per animal, each using 1 ml PBS/0.1 mM EDTA through 3-way stopcocks. Remove syringe from needle, inject recovered lavage fluid to 15 ml falcon tube on ice.
    Note: For lung tissue digestion, please go to step 22.
  10. Centrifuge microtubes containing ~1 ml BAL at 1,500 rpm for 5 min with brake.
  11. Pipet supernatant (BALF) from these tubes into fresh microtubes, store at -80 °C until ready to perform ELISA.
  12. Pipet 500 μl PBS to resuspend cell pellet in centrifuged microtubes and add all cells back into 15 ml conical tubes.
  13. Centrifuge 15 ml conical tubes at 1,500 rpm for 5 min with brake and resuspend cells with 1 ml erythrocyte lysis buffer and keep on ice for 5 min.
  14. Centrifuge the cells at 1,500 rpm for 5 min.
  15. Discard supernatant and resuspend BAL cells in 500 μl RPMI or FACS buffer.
  16. Add 50 μl of trypan blue to 50 μl saved aliquot, mix well and count cells using a hemocytometer. Calculate and record cell concentration.
    Note: Dark blue cells are dead and should not be counted. 
  17. Compute volume needed for 0.5-1 x 105 cells for each slide.
  18. Pre-wet cytospin funnels by spinning with 300 μl PBS onto glass slides (reusable) at 600 rpm for 5 min.
  19. Prepare microtubes containing 0.5-1 x 105 cells in 300 μl total volume. Spin BAL cells onto fresh labeled glass slides at 600 rpm for 10 min.
  20. Remove all slides from the cytospin apparatus, and allow to air dry at least 2 h.
  21. After dry, stain slides using Diff-Quick stain kit as follows:
    25 sec in fixative solution
    15 sec in solution I
    15 sec in solution II
    rinse the slide in distilled water.
  22. Perform differential cell counts under microscope at 100x magnification using oil-immersion lens (Figure 1).


    Figure 1. Photograph of cytospun BAL cells stained with Diff-Quick. (A) Control BAL; (B) BAL from asthmatic mice.

  23. The following protocol is for lung tissue digestion. Immediately after lavage, perfuse the lung vascular bed using a 10 ml syringe filled with 5 ml PBS. Make a small incision in the left ventricle and connect a 21 G needle and insert needle into the right ventricle. Accurate perfusion will result in a color change to white.
  24. Transfer lung lobes to a petri dish and chop it to small digestible pieces using a razor blade.
  25. Transfer grounded lung tissue into a 50 ml flask containing 20 ml/lung of tissue digestion solution and magnetic stir bar. Incubate, stirring at regular speed, at 37 °C for 30-45 min.
    Note: This can be performed in 37 °C incubator.
  26. Disperse the suspension by repeated aspiration through a 10 ml syringe, transfer to a 50 ml conical tube and centrifuge for 5 min at 1,500 rpm at 4 °C.
  27. Lyse remaining erythrocytes by suspension in erythrocyte lysis buffer for 2 min at room temperature. Wash cells with 10 ml cold PBS/0.5% BSA and centrifuge for 5 min at 1,500 rpm at 4 °C.
  28. Wash cells twice with 10 ml cold PBS/0.5% BSA, and filter through a 100-μm cell strainer.
  29. Resuspend 1 millions cells in 50 μl of 1:200 Fc block in FACS buffer and incubate for 10 min on ice.
  30. Wash the cells with 1 ml of PBS/0.5% BSA and spin down the cells for 5 min at 4 °C.
  31. Discard the supernatant and stain cells with antibodies (1:100 in FACS buffer) and incubate for 30 min on ice.
    Note: All the fluorochrome-conjugated mAbs were purchased from eBioscience or Biolegend.
  32. Wash the cells with 1 ml of PBS/0.5% BSA and spin down the cells for 5 min at 4 °C.
  33. Resuspend the cells in 500 μl PBS/0.5% BSA and analyze the cells using BD LSR II flow cytometer (Figure 2).


    Figure 2. Gating strategy for lung digested cells. This strategy also applies to BAL cells.

Recipes

  1. Erythrocyte lysis buffer
    NH4Cl 16.4 g
    KHCO3 2 g
    EDTA 0.5 M 400 μl
    2 L ddH2O
    Titrate with HCl to pH 7.2-7.4
  2. Tissue digestion solution
    Serum-free RPMI 1640
    0.13 mg/ml Liberase Blendzyme
    20 U/ml DNase

Acknowledgments

We thank the members of the Ziegler laboratory for discussion.

References

  1. Han, H., Headley, M. B., Xu, W., Comeau, M. R., Zhou, B. and Ziegler, S. F. (2013). Thymic stromal lymphopoietin amplifies the differentiation of alternatively activated macrophages. J Immunol 190(3): 904-912.

简介

支气管肺泡灌洗(BAL)是一种简单但有价值且通常执行的技术,通常用于研究肺病如哮喘和COPD的发病机制。 细胞计数可以与用于检查炎症反应的新方法组合,例如ELISA,流式细胞术分析,免疫组织化学,定量聚合酶链反应和HPLC以评估炎症细胞因子和生长因子的细胞表达。 在这里,我们描述了收集BAL液和消化肺组织以评估肺部成分数量的基本程序

关键字:鼠标, 肺功能, 肺疾病, 免疫细胞的功能, 黏膜免疫

材料和试剂

  1. 小鼠
  2. Avertin(Sigma-Aldrich,目录号:T48402)
  3. PBS(Sigma-Aldrich,目录号:D8537)
  4. RPMI-1640(Sigma-Aldrich,目录号:R8758)
  5. FACS缓冲液(PBS/0.5%BSA)
  6. Diff-Quick染色试剂盒(Dade Behring,目录号:B4132-1A)
  7. 单细胞学漏斗(Biomedical Polymers,Inc.,目录号:BMP-CYTO-S50)
  8. Superfrost载玻片(Thermo Fisher Scientific,目录号:22-034-979)
  9. Liberase Blendzyme(F.Hoffmann-La Roche,目录号:05401119001)
  10. DNase(F.Hoffmann-La Roche,目录号:10104159001)
  11. 台盼蓝(Life Technologies,Invitrogen TM ,目录号:15250-061)
  12. 小鼠Fc嵌段(BD Biosciences,Pharmingen TM ,目录号:553141)
  13. 用于T细胞的抗体(CD3 +
  14. 用于B细胞的抗体(B220 +
  15. 嗜酸性粒细胞抗体(Siglec-F + CD11c -
  16. 用于肺泡巨噬细胞的抗体(AMs,Siglec-F + CD11c CD11b int F4/80 +
  17. 间质巨噬细胞的抗体(Ims,Siglec-F -CD11c -CD11b + F4/80 +
  18. 中性粒细胞抗体(Siglec-F - CD11c - CD11b + Ly6G +
  19. 树突状细胞(DC)的抗体(Siglec-F -CD11c hi MHCII hi
  20. 红细胞裂解缓冲液(参见配方)
  21. 组织消化溶液(参见配方)

设备

  1. 1 ml,3 ml和10 ml无菌注射器
  2. 21号灌洗管
  3. 21号无菌针
  4. 棉线40号
  5. 50ml烧瓶中
  6. 15 ml和50 ml锥形管
  7. 微管
  8. 100微米细胞滤器(BD Biosciences,Falcon ,目录号:352360)
  9. 血细胞计数器
  10. 离心机(Beckman Coulter,型号:Allegra TM 6R)
  11. Cytospin细胞离心机(Thermo Fisher Scientific/Shandon,型号:A7830002)
  12. 显微镜
  13. BD LSR II流式细胞仪
  14. 3-way Stopcocks(Bio-Rad Laboratories,型号:732-8103)
  15. 磁力搅拌棒(VWR International,型号:58949-006)
  16. 多位置磁力搅拌器(VWR International,型号:12621-042)

程序

  1. 通过腹膜内注射1ml 2.5%Avertin的PBS麻醉小鼠
  2. 使用剪刀暴露胸腔和颈部。 解剖组织从颈部暴露气管
  3. 继续打开隔膜切割肋骨,暴露心脏和肺。 小心不要刺破心脏或肺。
  4. 使用镊子在气管下滑动1英寸长的螺纹
  5. 在气管中做一个小切口,允许21规格的灌洗管通过气管。气管近端与气管切口之间的距离为2-3mm。
    注意:不要一直割伤气管。
  6. 切割21号管的1-1.5英寸部分,小心地将21号针穿入管中。将管插入气管,并将管线缠绕在气管中的管周围
  7. 使用"输入"3毫升注射器通过三通旋塞,缓慢注射1毫升冷PBS的0.1毫米EDTA到肺中:注意肺膨胀,不要过度充气。
  8. 收集约1毫升BAL液(BALF)从肺,使用"输出"3毫升注射器在冰上的微管中。
  9. 重复步骤7-8每个动物3次洗涤,每次使用1ml PBS/0.1mM EDTA通过3通活塞。从针头取出注射器,注射回收的灌洗液在冰上15毫升falcon管 注意:对于肺组织消化,请转到第22步。
  10. 离心含有〜1 ml BAL的微管,在1,500 rpm下制动5分钟。
  11. 吸管上清液(BALF)从这些管放入新鲜的微管,存储在-80°C,直到准备好进行ELISA。
  12. 吸取500微升PBS重悬细胞沉淀在离心微管,并添加所有细胞回到15毫升锥形管。
  13. 离心15毫升锥形管在1,500 rpm 5分钟与制动和重悬细胞与1毫升红细胞裂解缓冲液,并保持在冰上5分钟。
  14. 以1,500 rpm离心细胞5分钟。
  15. 弃去上清液并将BAL细胞重悬于500μlRPMI或FACS缓冲液中
  16. 加入50微升台盼蓝到50微升保存的等分试样,混匀,使用血细胞计数细胞。 计算并记录细胞浓度。
  17. 计算每个幻灯片0.5-1 x 10 5 个单元所需的体积
  18. 通过用300μlPBS旋转在玻璃载玻片(可重复使用)上以600rpm离心5分钟预湿润细胞离心涂布漏斗。
  19. 准备含有0.5-1×10 5个细胞的微量管,总体积为300μl。 旋转BAL细胞到新鲜的标签玻璃载玻片上,在600 rpm下10分钟
  20. 从细胞离心涂片装置中取出所有载玻片,并允许空气干燥至少2小时。
  21. 干燥后,使用Diff-Quick染色试剂盒染色载玻片,如下:
    25秒固定溶液
    在溶液I中为15秒
    溶液II中为15秒
    用蒸馏水冲洗载玻片。
  22. 使用油浸镜在100倍放大倍数下在显微镜下进行微分细胞计数(图1)

    图1.用Diff-Quick染色的cytospun BAL细胞的照片。(A)对照BAL; (B)来自哮喘小鼠的BAL。

  23. 以下方案用于肺组织消化。 在灌洗后,立即使用填充有5ml PBS的10ml注射器灌注肺血管床。 在左心室做一个小切口,并连接一个21 G针和插入针到右心室。 精确灌注会导致颜色变为白色。
  24. 转移肺叶到培养皿,并使用剃刀切割成小的可消化的碎片。
  25. 将接地的肺组织转移到含有20ml /肺组织消化溶液和磁力搅拌棒的50ml烧瓶中。 孵育,以常规速度,在37℃下搅拌30-45分钟。
    注意:这可以在37℃的培养箱中进行。
  26. 通过重复抽吸通过10ml注射器分散悬浮液,转移到50ml锥形管中,并在4℃下以1,500rpm离心5分钟。
  27. 通过悬浮在红细胞裂解缓冲液中在室温下裂解剩余的红细胞2分钟。 用10ml冷PBS/0.5%BSA洗涤细胞并在4℃以1,500rpm离心5分钟。
  28. 用10ml冷PBS/0.5%BSA洗涤细胞两次,并通过100μm细胞过滤器过滤。
  29. 在FACS缓冲液中在50μl1:200Fc块中重悬1百万个细胞,并在冰上孵育10分钟。
  30. 用1ml PBS/0.5%BSA洗涤细胞,并在4℃下旋转细胞5分钟
  31. 用抗体(在FACS缓冲液中1:100)弃去上清液和染色细胞,并在冰上孵育30分钟。
    注意:所有荧光染料偶联的mAb购自eBioscience或Biolegend。
  32. 用1ml PBS/0.5%BSA洗涤细胞,并在4℃下旋转细胞5分钟
  33. 重悬细胞在500μlPBS/0.5%BSA,并使用BD LSR II流式细胞仪分析细胞(图2)。


    图2.肺消化细胞的门控策略。 此策略也适用于BAL单元格。

食谱

  1. 红细胞裂解缓冲液
    NH 4 Cl 16.4g
    KHCO 3 2 g
    EDTA 0.5M400μl
    2 L ddH 2 O 2/b 用HCl滴定至pH 7.2-7.4
  2. 组织消化溶液
    无血清RPMI 1640
    0.13mg/ml Liberase Blendzyme
    20 U/ml DNase

致谢

我们感谢齐格勒实验室的成员进行讨论。

参考文献

  1. Han,H.,Headley,M.B.,Xu,W.,Comeau,M.R.,Zhou,B.and Ziegler,S.F。 胸腺基质淋巴细胞生成素放大交替激活的巨噬细胞的分化。/em> 190(3):904-912。
  • English
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
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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