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Isolation of Lung Infiltrating Cell in Mice
小鼠肺部免疫浸润细胞的分离   

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Abstract

Inflammatory lung diseases induce strong leukocyte recruitment into the organ, culminating in pneumonia area formation. Here, we describe the protocol for isolation of lung infiltrating cells. Using this assay, we analyzed the lung cell phenotyping by flow cytometry and spontaneous cytokine production by cultivating lung cells ex vivo (Amaral et al., 2014).

Keywords: Tuberculosis(肺结核), Inflammation(炎症), Lung cells(肺细胞), Isolation(隔离), Single cell(单细胞)

Materials and Reagents

  1. Mice
  2. Collagenase Type IV (0.5 mg/ml) (Sigma-Aldrich, catalog number: C5138 ) or Liberase Blendzyme 2 (2 μg/ml) (Roche Diagnostics, catalog number: 1988433 )
  3. RPMI 1640 (Life Technologies, catalog number: 11875-093 )
  4. Gentamicin (10 μg/ml) (Life Technologies, catalog number: 15750-102 )
  5. Type IV DNase I from bovine pancreas (25 units/ml) (Sigma-Aldrich, catalog number: D5025 ) or DNase I from bovine pancreas (25 units/ml) (Roche Diagnostics, catalog number: 11284932001 )
  6. Fetal bovine serum heat inactivated (Life Technologies, catalog number: 10437-028 )
  7. Red cells lysing buffer (see Recipes)
  8. 1x phosphate buffered saline (PBS) (see Recipes)
  9. 2x digestion medium (see Recipes)

Equipment

  1. Syringes (BD Biosciences, catalog number: 301604 )
  2. Centrifuge 5810 (Eppendorf)
  3. 18-gauge needle (BD Biosciences, catalog number: 305180 )
  4. Cell strainer (100-μm pore size) (Corning, catalog number: 352360 )
  5. Sterile culture hood

Procedure

  1. The lungs are harvested after animal euthanasia. Sterilize the skin of the mice with 70% ethanol and cut the skin and ribs using scissors to expose the thoracic cavity.
  2. Remove the left lung lobes.
  3. Wash the lungs with sterile 1x PBS and placed in Petri dishes with RPMI 1640 medium* (2 ml).
  4. Mince organ with forceps and scissors to 1mm sized chunks.
  5. Once the lung dissected, incubate the tissue with 2 ml of 2x digestion medium* (final concentration: 0.5 mg/ml) or liberase (final concentration 2 μg/ml) and Type IV DNase I (final concentration: 25 units/ml) at 37 °C under agitation (200 rpm) conditions for 45 min.
    *Note: If use the whole lung, it is recommended mince organs in 3 ml of RPMI 1640 medium and then add 3 ml of 2x digestion medium.
  6. Stop the reaction adding 1 ml of FBS heat inactivated.
  7. Disperse the cells with a 10-ml syringe fitted with an 18-gauge needle (10-times).
  8. Filter the cells using cell strainer (100 μm) using 50 ml conical tube to remove tissue debris. Transfer the homogenate from 50 ml to 15 ml conical tube prior centrifugation.
  9. Centrifuge the cells at 10 °C and 300 x g for 5 min.
  10. Remove the supernatant by aspirating.
  11. Add 1 ml of Red cells lysing buffer and incubate at room temperature for 1 min.
  12. Stop the reaction adding 10 ml of 1x PBS with 10% FBS heat inactivated.
  13. Centrifuge the cells at 10 °C and 300 x g for 5 min.
  14. Discard the supernatant and resuspend the pellet in RPMI supplemented with 10% FBS and gentamicin (10 μg/ml). Cells have to be maintained in the fridge prior to flow cytometry processing or ex vivo pulmonary cell cultures.

Recipes

  1. Red cells lysing buffer
    0.144 M NH4Cl
    0.0169 M TRIS base
    pH 7.4
    Note: Adjust the pH using HCl and NaOH.
  2. 1x phosphate buffered saline (PBS)
    Dissolve the following in 800ml distilled H2O
    8 g of NaCl
    0.2 g of KCl
    1.44g of Na2HPO4
    0.24 g of KH2PO4
    Adjust pH to 7.4
    Adjust volume to 1L with additional distilled H2O
    Sterilize the solution
    Note: Adjust the pH using HCl and NaOH.
  3. 2x digestion medium
    2 mg of Collagenase type IV or 4 μg/ml of liberase
    50 units of type IV DNase I from bovine pancreas
    2 ml of RPMI 1640 medium

Acknowledgments

This work was supported by an award from FAPESP (number: 2010/19246-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Amaral, E. P., Ribeiro, S. C., Lanes, V. R., Almeida, F. M., de Andrade, M. R., Bomfim, C. C., Salles, E. M., Bortoluci, K. R., Coutinho-Silva, R., Hirata, M. H., Alvarez, J. M., Lasunskaia, E. B. and D'Imperio-Lima, M. R. (2014). Pulmonary infection with hypervirulent Mycobacteria reveals a crucial role for the P2X7 receptor in aggressive forms of tuberculosis. PLoS Pathog 10(7): e1004188.

简介

炎性肺疾病诱导强烈的白细胞募集到器官中,最终导致肺炎区域形成。 在这里,我们描述的隔离肺浸润细胞的协议。 使用该测定,我们通过流式细胞术和通过离体培养肺细胞的自发细胞因子产生来分析肺细胞表型(Amaral等人,2014)。

关键字:肺结核, 炎症, 肺细胞, 隔离, 单细胞

材料和试剂

  1. 小鼠
  2. 胶原酶IV型(0.5mg/ml)(Sigma-Aldrich,目录号:C5138)或Liberase Blendzyme 2(2μg/ml)(Roche Diagnostics,目录号:1988433)
  3. RPMI 1640(Life Technologies,目录号:11875-093)
  4. 庆大霉素(10μg/ml)(Life Technologies,目录号:15750-102)
  5. 来自牛胰腺(25单位/ml)(Sigma-Aldrich,目录号:D5025)或来自牛胰腺(25单位/ml)的DNase I的IV型DNase I(Roche Diagnostics,目录号:11284932001)
  6. 胎牛血清热灭活(Life Technologies,目录号:10437-028)
  7. 红细胞裂解缓冲液(见配方)
  8. 1×磷酸盐缓冲盐水(PBS)(见Recipes)
  9. 2x消化培养基(参见配方)

设备

  1. Syringes(BD Biosciences,目录号:301604)
  2. 离心机5810(Eppendorf)
  3. 18号针(BD Biosciences,目录号:305180)
  4. 细胞过滤器(100μm孔径)(Corning,目录号:352360)
  5. 无菌培养罩

程序

  1. 在动物安乐死后收获肺。 消毒小鼠的皮肤用70%乙醇和切割皮肤和肋骨,使用剪刀暴露胸腔。
  2. 取出左肺叶。
  3. 用无菌1x PBS洗涤肺,并置于含有RPMI 1640培养基*(2ml)的培养皿中
  4. 剁碎器官用钳子和剪刀到1mm大小的块
  5. 一旦肺解剖,用2ml 2×消化培养基*(终浓度:0.5mg/ml)或释放酶(终浓度2μg/ml)和IV型DNA酶I(终浓度:25单位/ml) 37℃,搅拌(200rpm)条件下45分钟 注意:如果使用全肺,建议在3ml RPMI 1640培养基中加入3ml 2×消化培养基。
  6. 停止反应,加入1ml FBS热灭活的
  7. 用装有18号针头的10-ml注射器(10次)分散细胞
  8. 使用细胞过滤器(100μm)使用50ml锥形管除去组织碎片过滤细胞。 在离心前将匀浆从50ml至15ml锥形管转移
  9. 在10℃和300×g离心细胞5分钟
  10. 通过吸气除去上清液。
  11. 加入1ml红细胞裂解缓冲液,并在室温下孵育1分钟
  12. 停止反应,加入10 ml含10%FBS的1×PBS热灭活
  13. 在10℃和300×g离心细胞5分钟
  14. 弃去上清液,并在补充有10%FBS和庆大霉素(10μg/ml)的RPMI中重悬沉淀。 在流式细胞术处理或离体肺细胞培养之前,细胞必须保持在冰箱中

食谱

  1. 红细胞裂解缓冲液
    0.144 M NH 4 Cl·
    0.0169 M TRIS基本
    pH 7.4
    注意:使用HCl和NaOH调整pH值。
  2. 1×磷酸盐缓冲盐水(PBS)
    将以下物质溶于800ml蒸馏的H 2 O中 8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24g的KH 2 PO 4 sub/
    将pH调节至7.4
    用另外的蒸馏H 2 O 2调节体积至1L 消毒溶液
    注意:使用HCl和NaOH调整pH值。
  3. 2x消化培养基
    2mg胶原酶IV型或4μg/ml释放酶
    50单位来自牛胰腺的IV型DNase I 2ml RPMI 1640培养基

致谢

这项工作得到了FAPESP奖(数字:2010/19246-1)的支持。 资助者在研究设计,数据收集和分析,决定发布或准备手稿方面没有任何作用。

参考文献

  1. Amaral,EP,Ribeiro,SC,Lane,VR,Almeida,FM,de Andrade,MR,Bomfim,CC,Salles,EM,Bortoluci,KR,Coutinho-Silva,R.,Hirata,MH,Alvarez,JM,Lasunskaia, EB和D'Imperio-Lima,MR(2014)。 肺部感染的高毒性分枝杆菌揭示了P2X7受体的关键作用 攻击性形式的结核病。 PLoS Pathog 10(7):e1004188。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Amaral, E. P., Lassunskaia, E. B. and D’Império-Lima, M. R. (2015). Isolation of Lung Infiltrating Cell in Mice . Bio-protocol 5(4): e1403. DOI: 10.21769/BioProtoc.1403.
  2. Amaral, E. P., Ribeiro, S. C., Lanes, V. R., Almeida, F. M., de Andrade, M. R., Bomfim, C. C., Salles, E. M., Bortoluci, K. R., Coutinho-Silva, R., Hirata, M. H., Alvarez, J. M., Lasunskaia, E. B. and D'Imperio-Lima, M. R. (2014). Pulmonary infection with hypervirulent Mycobacteria reveals a crucial role for the P2X7 receptor in aggressive forms of tuberculosis. PLoS Pathog 10(7): e1004188.
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