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Murine Bronchoalveolar Lavage
小鼠支气管肺泡灌洗   

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参见作者原研究论文

本实验方案简略版
Oncogene
May 2016

Abstract

A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, etc., to examine the immune cells, pathogens, proteins such as cytokines/chemokines, and the expression levels of inflammation-related and other genes in the cells. This will help to understand the underlying mechanisms of these lung diseases and develop specific and effective drugs.

Keywords: Bronchoalveolar lavage (支气管肺泡灌洗), Lung cancer (肺癌), Hema3-staining (Hema3染色)

Background

Bronchoalveolar lavage (BAL) is a simple and typical method commonly performed to diagnose pulmonary diseases including lung cancer (Daubeuf and Frossard, 2012). It is used to sample pulmonary components to determine the protein composition, immune cells and pathogens in the lung. Pulmonary chronic inflammation plays a critical role in lung cancer initiation and progression. To clarify the underlying mechanism of inflammation in lung tumorigenesis, a basic BAL protocol in mice is used in our laboratory to determine the pulmonary immune response (Qu et al., 2015; Zhou et al., 2015; Sun et al., 2016; Zhou et al., 2017).

Materials and Reagents

  1. Needles (BD, catalog number: 305167 ) or tapes
  2. Nylon string (Dynarex, catalog number: 3243 )
  3. 22 G x 1” Exel Safelet Catheter (Exel International, catalog number: 26746 )
  4. 1 ml syringe (BD, catalog number: 309659 )
  5. 1.5 ml Eppendorf tubes (VWR, catalog number: 87003-294 )
  6. 0.22 µm filter (EMD Millipore, catalog number: SLGP033RS )
  7. Mice (THE JACKSON LABORATORY)
  8. 70% ethanol (Decon Labs, catalog number: 2701 )
  9. Protease inhibitor cocktail (Roche Diagnostics, catalog number: 11697498001 )
  10. Phenylmethylsulfonyl fluoride (PMSF) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 36978 )
  11. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
  12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  13. Disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: BP331-500 )
  14. Potassium phosphate monobasic (KH2PO4) (Acros Organics, catalog number: 205925000 )
  15. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434-1KG )
  16. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: P9144-1KG )
    Note: This product has been discontinued.
  17. Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2-EDTA-2H2O) (Sigma-Aldrich, catalog number: E5134-100G )
  18. Phosphate-buffered saline (PBS) (see Recipes)
  19. ACK lysis buffer (see Recipes)

Equipment

  1. CO2 chamber
  2. Biosafety cabinet
  3. Styrofoam board
  4. Forceps (Roboz Surgical Instrument, catalog number: RS-5135 )
  5. Scissors (Roboz Surgical Instrument, catalog number: RS-6802 )
  6. Centrifuge (Eppendorf, model: 5417 R )
  7. Hemocytometer (Hausser Scientific, catalog number: 3110 )
  8. Microscope (Olympus, model: CK30 )

Procedure

  1. Sacrifice a mouse by CO2 inhalation in a CO2 chamber.
  2. Bring the mouse to a biosafety cabinet.
  3. Dampen the mouse with 70% ethanol.
  4. Place the mouse front side up on styrofoam panel and fix the arms and legs with needles or tapes (Figure 1A).
  5. Use scissors to make incision in the skin from abdomen to neck, and tear skin with forceps to expose thoracic cage and neck (Figure 1A).
  6. Gently remove the muscle around the neck to expose trachea (Figure 1B).
  7. Use forceps to put a ~10 cm-long nylon string under the trachea (Figure 1C).
  8. Carefully cut the ribs to expose the heart and the lungs (Figure 1D).
    Note: Do not cut the trachea and lungs.
  9. Insert a 22 G x 1” Exel Safelet Catheter into the trachea, remove the stylet hub, and tie the catheter and the trachea together firmly with a nylon string (Figures 1E-1G).
  10. Load a 1 ml syringe with 0.8 ml of PBS and place it in the end of the catheter (Figure 1H).


    Figure 1. Mouse bronchoalveolar lavage steps

  11. Slowly inject and aspirate the PBS 4 times, remove the syringe from catheter, and save the recovered lavage fluid in 1.5 ml Eppendorf (EP) tubes on ice.
  12. Repeat steps 10-11 for additional three times.
  13. Record BAL volume according to the scales on the 1.5 ml EP tubes.
  14. Centrifuge at 800 x g for 10 min at 4 °C.
  15. Transfer the supernatant to a new 5 ml tube, add protease inhibitor cocktail to a final concentration of 1x and PMSF to a final concentration of 1 mM, and mix well. Store at -80 °C if not used immediately. Pathogens, proteins such as cytokines/chemokines, and other components in the supernatants of BAL fluid (BALF) can be analyzed by bicinchoninic acid protein assay, ELISA, luminex assays, and/or other assays.
  16. Resuspend BAL cell pellets in 200 µl of PBS and combine the cell suspensions.
  17. Centrifuge at 800 x g for 10 min at 4 °C.
  18. Resuspend BAL cell pellets in 100-200 µl ACK lysis buffer, lyse red blood cells on ice for 10 min, and then add 1 ml of PBS to stop lysis.
  19. Centrifuge at 800 x g for 10 min at 4 °C.
  20. Resuspend BAL cell pellets in 400 µl of PBS, count the cells by taking about 20 µl of the cell sample to a hemocytometer and counting the cells under a microscope. Record the cell number. These BAL cells can then be used for FACS analysis, spun onto slides for staining (Figure 2), and/or lysed for RNA or protein extraction.


    Figure 2. Hema3-staining of immune cells in murine BAL. White arrow: macrophage; grey arrow: lymphocyte; black arrow: neutrophil. Scale bar = 10 µm.

Data analysis

Murine BAL cells are retrieved and cytospun onto slides, which are Hema3-stained to determine the immune cells.

Notes

Although pelleted BAL cells from all four washes are combined to analyze the cells, you can combine the supernatant from only the first two washes (Procedure step 11) to analyze the BAL fluid components, as the first two washes contain a high concentration of these components.

Recipes

  1. Phosphate-buffered saline (PBS) (1 L)
    8.0 g NaCl
    0.2 g KCl
    1.15 g Na2HPO4·7H2O
    0.2 g KH2PO4
    Adjust to pH 7.4
  2. ACK lysis buffer (1 L)
    1 L deionized water
    8.29 g NH4Cl
    1 g KHCO3
    37.2 mg Na2-EDTA
    Adjust pH to 7.2-7.4, sterilize using 0.22 µm filter, store at 4 °C

Acknowledgments

The authors thank the members in Xiao-Qu lab for the helpful discussion. This study was supported in part by the National Institute of Health (NIH)/National Cancer Institute (NCI) grants R01 CA172090, R21 CA175252, P50 CA090440, P30 CA047904, as well as the American Lung Association (ALA) Lung Cancer Discovery Award LCD 259111 and American Cancer Society (ACS) Fellowship Award PF-12081-01-TBG.

References

  1. Daubeuf, F. and Frossard, N. (2012). Performing bronchoalveolar lavage in the mouse. Curr Protoc Mouse Biol 2(2):167-175.
  2. Qu, Z., Sun, F., Zhou, J., Li, L., Shapiro, S. D. and Xiao, G. (2015). Interleukin-6 prevents the initiation but enhances the progression of lung cancer. Cancer Res 75(16): 3209-3215.
  3. Sun, F., Qu, Z., Xiao, Y., Zhou, J., Burns, T. F., Stabile, L. P., Siegfried, J. M. and Xiao, G. (2016). NF-κB1 p105 suppresses lung tumorigenesis through the Tpl2 kinase but independently of its NF-κB function. Oncogene 35(18): 2299-2310.
  4. Zhou, J., Qu, Z., Sun, F., Han, L., Li, L., Yan, S., Stabile, L. P., Chen, L. F., Siegfried, J. M. and Xiao, G. (2017). Myeloid STAT3 promotes lung tumorigenesis by transforming tumor immunosurveillance into tumor-promoting inflammation. Cancer Immunol Res 5(3): 257-268.
  5. Zhou, J., Qu, Z., Yan, S., Sun, F., Whitsett, J. A., Shapiro, S. D. and Xiao, G. (2015). Differential roles of STAT3 in the initiation and growth of lung cancer. Oncogene 34(29): 3804-3814.

简介

这里描述了小鼠中基本的支气管肺泡灌洗(BAL)程序。可以通过Hema3染色,免疫染色,荧光激活细胞分选(FACS),PCR,二金鸡宁酸蛋白测定,酶联免疫吸附测定(ELISA),luminex检测等来分析从BAL获得的细胞和液体。 / em>,以检查免疫细胞,病原体,蛋白质如细胞因子/趋化因子,以及细胞中炎症相关基因和其他基因的表达水平。这将有助于了解这些肺部疾病的潜在机制,并开发具体有效的药物。

背景 支气管肺泡灌洗(BAL)是通常用于诊断肺部疾病(包括肺癌)的简单且典型的方法(Daubeuf和Frossard,2012)。它用于采样肺组分,以确定肺中的蛋白质组成,免疫细胞和病原体。肺部慢性炎症在肺癌起始和进展中起关键作用。为了阐明肺肿瘤发生的炎症的潜在机制,我们的实验室使用了一种基本的BAL方案来确定肺部免疫反应(Qu等人,2015; Zhou等)。 ,2015; Sun等人,2016; Zhou等人,2017)。

关键字:支气管肺泡灌洗, 肺癌, Hema3染色

材料和试剂

  1. 针(BD,目录号:305167)或磁带
  2. 尼龙绳(Dynarex,目录号:3243)
  3. 22 G x 1"Exel Safelet导管(Exel International,目录号:26746)
  4. 1 ml注射器(BD,目录号:309659)
  5. 1.5ml Eppendorf管(VWR,目录号:87003-294)
  6. 0.22μm过滤器(EMD Millipore,目录号:SLGP033RS)
  7. 小鼠(杰克逊实验室)
  8. 70%乙醇(Decon Labs,目录号:2701)
  9. 蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11697498001)
  10. 苯甲基磺酰氟(PMSF)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:36978)
  11. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9625)
  12. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  13. 七水合磷酸氢二钠(Na 2 HPO 4·7H 2 O)(Fisher Scientific,目录号:BP331-500)
  14. 磷酸二氢钾(KH 2 PO 4)(Acros Organics,目录号:205925000)
  15. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A9434-1KG)
  16. 碳酸氢钾(KHCO 3)(Sigma-Aldrich,目录号:P9144-1KG)
    注意:本产品已停产。
  17. 乙二胺四乙酸二钠盐二水合物(Na 2 O 2 -EDTA-2H 2 O)(Sigma-Aldrich,目录号:E5134-100G)
  18. 磷酸盐缓冲盐水(PBS)(见食谱)
  19. ACK裂解缓冲液(参见食谱)

设备

  1. CO 2
  2. 生物安全柜
  3. 泡沫塑料板
  4. 镊子(Roboz手术器械,目录号:RS-5135)
  5. 剪刀(Roboz手术器械,目录号:RS-6802)
  6. 离心机(Eppendorf,型号:5417 R)
  7. 血细胞计数器(Hausser Scientific,目录号:3110)
  8. 显微镜(Olympus,型号:CK30)

程序

  1. 在CO 2室中通过CO 2吸入牺牲小鼠。
  2. 将鼠标移到生物安全柜。
  3. 用70%乙醇减压老鼠。
  4. 将鼠标放在聚苯乙烯泡沫面板上,用手指或胶带固定手臂和腿部(图1A)。
  5. 使用剪刀从腹部到颈部切开皮肤,用镊子撕开皮肤以暴露胸廓和颈部(图1A)。
  6. 轻轻取出颈部肌肉以暴露气管(图1B)
  7. 使用镊子将〜10厘米长的尼龙线放在气管下(图1C)。
  8. 仔细切开肋骨以暴露心脏和肺部(图1D)。
    注意:不要切断气管和肺。
  9. 将22 G x 1"Exel Safelet导管插入气管,取出探针枢纽,并用尼龙线将导管和气管连接牢固(图1E-1G)。
  10. 加入1 ml注射器,加入0.8 ml PBS,将其置于导管末端(图1H)

    图1.小鼠支气管肺泡灌洗步骤

  11. 缓慢注射和吸取PBS 4次,从导管中取出注射器,并将回收的灌洗液在冰上保存在1.5ml Eppendorf(EP)管中。
  12. 重复步骤10-11再三次。
  13. 根据1.5 ml EP管上的刻度记录BAL体积。
  14. 在4℃下以800 x g离心10分钟。
  15. 将上清液转移到新的5ml管中,加入蛋白酶抑制剂混合物至终浓度为1mM和PMSF至终浓度为1mM,并充分混合。储存于-80°C,如果不立即使用。 BAL液(BALF)上清液中的细胞因子/趋化因子和其他成分的蛋白质可以通过二辛可宁酸蛋白测定法,ELISA,luminex测定法和/或其他测定法进行分析。
  16. 将BAL细胞沉淀重悬于200μlPBS中,并结合细胞悬液
  17. 在4℃下以800 x g离心10分钟。
  18. 将BAL细胞沉淀重悬于100-200μlACK裂解缓冲液中,在冰上裂解红细胞10分钟,然后加入1ml PBS以停止裂解。
  19. 在4℃下以800 x g离心10分钟。
  20. 将BAL细胞沉淀重悬于400μlPBS中,通过将约20μl的细胞样品摄入血细胞计数器并在显微镜下计数细胞来计数细胞。记录单元格编号。然后将这些BAL细胞用于FACS分析,旋转到载玻片上进行染色(图2),和/或裂解用于RNA或蛋白质提取。


    图2.鼠BAL中免疫细胞的Hema3染色。白色箭头:巨噬细胞;灰色箭头:淋巴细胞;黑色箭头:嗜中性粒细胞。比例尺= 10μm

数据分析

取出小鼠BAL细胞,并将细胞移植到载玻片上,Hema3染色以确定免疫细胞。

笔记

虽然来自所有四个洗涤液的沉淀的BAL细胞被组合以分析细胞,但是可以将来自前两次洗涤的上清液(方法步骤11)组合以分析BAL液体组分,因为前两次洗涤含有这些组分的高浓度。

食谱

  1. 磷酸盐缓冲盐水(PBS)(1升)
    8.0克NaCl
    0.2克KCl
    1.15g Na 2 HPO 4< 7H 2< 2><< / 0.2g KH 2 PO 4
    调整至pH 7.4
  2. ACK裂解缓冲液(1L)
    1升去离子水
    8.29g NH 4 Cl
    1克KHCO 3
    37.2mg Na 2 -EDTA
    将pH调节至7.2-7.4,使用0.22μm过滤器灭菌,在4℃下储存

致谢

作者感谢小曲实验室的成员进行了有益的讨论。这项研究部分由国立卫生研究院(NIH)/国家癌症研究所(NCI)授权R01 CA172090,R21 CA175252,P50 CA090440,P30 CA047904以及美国肺协会(ALA)肺癌发现奖LCD 259111和美国癌症协会(ACS)奖学金奖PF-12081-01-TBG。

参考

  1. Daubeuf,F。和Frossard,N.(2012)。在小鼠中进行支气管肺泡灌洗。 Curr Protoc Mouse Biol 2(2):167-175。
  2. Qu,Z.,Sun,F.,Zhou,J.,Li,L.,Shapiro,SD and Xiao,G.(2015)。  白细胞介素-6阻止起始,但可以增强肺癌的发展。癌症研究 75(16 ):3209-3215。
  3. Sun,F.,Qu,Z.,Xiao,Y.,Zhou,J.,Burns,TF,Stabile,LP,Siegfried,JM和Xiao,G。(2016)。 NF-κB1p105通过Tpl2激酶抑制肺部肿瘤发生,但与NF-κB功能无关。 a>癌基因 35(18):2299-2310。
  4. Zhou,J.,Qu,Z.,Sun,F.,Han,L.,Li,L.,Yan,S.,Stabile,LP,Chen,LF,Siegfried,JM和Xiao,G。(2017)。   髓系STAT3通过将肿瘤免疫监视转化为肿瘤免疫监测来促进肺部肿瘤发生,促进炎症。癌症免疫组织研究 5(3):257-268。
  5. Zhou,J.,Qu,Z.,Yan,S.,Sun,F.,Whitsett,JA,Shapiro,SD和Xiao,G。(2015)。 STAT3在肺癌的起始和生长中的差异作用。癌基因 34 (29):3804-3814。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sun, F., Xiao, G. and Qu, Z. (2017). Murine Bronchoalveolar Lavage. Bio-protocol 7(10): e2287. DOI: 10.21769/BioProtoc.2287.
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