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Group 2 innate lymphoid cells (ILC2) are a recently characterized cell population which lacks specific antigen receptors and contributes to immune responses at mucosal surfaces of lung and gut. Recently, we demonstrated that ILC2 expand in the context of chronic liver diseases in mice and contribute to pathology in an IL-33 dependent manner. Here, we describe a protocol to isolate highly purified ILC2 from mouse livers. This procedure provides effective digestion of liver tissue and limits proteolytic degradation of cell surface receptors leading to increased yields of biologically functional cells. The number of liver resident ILC2 in the steady state is low, however their number dramatically increase upon systemic or local treatment of mice with cytokines such as IL-25 and IL-33. Using minicircle-based expression constructs for these cytokines high numbers of functional ILC2 can be isolated with the protocol provided here.

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Isolation of ILC2 from Mouse Liver
从小鼠肝脏中分离第2组先天淋巴细胞(ILC2)

免疫学 > 免疫细胞分离 > 淋巴细胞
作者: Tamar Mchedlidze
Tamar MchedlidzeAffiliation: Medical Department 1, FAU Erlangen-Nuremberg, Erlangen, Germany
Bio-protocol author page: a1516
 and Stefan Wirtz
Stefan WirtzAffiliation: Medical Department 1, FAU Erlangen-Nuremberg, Erlangen, Germany
For correspondence: stefan.wirtz@uk-erlangen.de
Bio-protocol author page: a1517
Vol 4, Iss 14, 7/20/2014, 3758 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1179

[Abstract] Group 2 innate lymphoid cells (ILC2) are a recently characterized cell population which lacks specific antigen receptors and contributes to immune responses at mucosal surfaces of lung and gut. Recently, we demonstrated that ILC2 expand in the context of chronic liver diseases in mice and contribute to pathology in an IL-33 dependent manner. Here, we describe a protocol to isolate highly purified ILC2 from mouse livers. This procedure provides effective digestion of liver tissue and limits proteolytic degradation of cell surface receptors leading to increased yields of biologically functional cells. The number of liver resident ILC2 in the steady state is low, however their number dramatically increase upon systemic or local treatment of mice with cytokines such as IL-25 and IL-33. Using minicircle-based expression constructs for these cytokines high numbers of functional ILC2 can be isolated with the protocol provided here.
Keywords: Group 2 innate lymphoid cells(集团2细胞感染的研究), ILC2(I LC2), Inflammation(炎症)

[Abstract]

Materials and Reagents

  1. Liver from a IL-33 or IL-25 treated mouse (e.g. C57BL/6 or Balb/c)
  2. Fetal calf serum (FCS)
  3. RPMI-1640 medium
  4. Easycoll separation solution (density 1.124) (Biochrom, catalog number: L6145 )
  5. Anti-mouse CD16/CD32 (Fc-Block) (eBioscience, catalog number: 14-0161 )
  6. 0.5 M CaCl2 solution
  7. 0.2 M MgCl2 solution
  8. AutoMACS rinsing solution (Miltenyi Biotec)
  9. Brilliant Violet 421 Streptavidin (BioLegend, catalog number: 405226 )
  10. Antibodies
    1. FACS antibodies
      1. Anti-Sca-1 (clone D7, FITC conjugated) (eBioscience, catalog number: 11-5981 )
      2. Anti-KLRG1 (clone 2F1, APC conjugated) (eBioscience, catalog number: 17-5893 )
      3. Anti-ICOS (clone 7E.17G9, PE conjugated) (eBioscience, catalog number: 12-9942 )
    2. Anti-mouse lineage-antibodies biotin labelled
      1. Anti-CD3 (clone 145-2C11) (eBioscience, catalog number: 13-0031 )
      2. Anti-CD45R (clone RA3-6B2) (eBioscience, catalog number: 13-0452 )
      3. Anti-Ly-6G (clone RB6-8C5) (eBioscience, catalog number: 13-5931 )
      4. Anti-CD11b (clone M1/70) (eBioscience, catalog number: 13-0112 )
      5. Anti-NK1.1 (clone PK136) (eBioscience, catalog number: 13-5941 )
      6. Anti-Ter-119 (clone Ter-119) (eBioscience, catalog number: 13-5921 )
      7. Anti-SiglecF (clone ES22-10D8) (Miltenyi Biotec, catalog number: 130-101-861 )
      8. Anti-CD5 (clone 53-7.3) (Miltenyi Biotec, catalog number: 130-101-960 )
  11. Krebs-Ringer-Buffer (KRB) (see Recipes)
  12. Collagenase IV solution (Sigma-Aldrich, catalog number: C5138 ) (see Recipes)
  13. DNase I solution (Roche Diagnostics, catalog number: 10104159001 ) (see Recipes)
  14. 30% Biocoll (see Recipes)
  15. 80% Biocoll (see Recipes)
  16. FACS buffer (see Recipes)
  17. ACK buffer (see Recipes)
  18. PEB buffer (see Recipes)

Equipment

  1. GentleMACS C tube (Miltenyi Biotec)
  2. 6 well plates
  3. GentleMACS dissociator (Miltenyi Biotec)
  4. MACSmix tube rotator (Miltenyi Biotec)
  5. Centrifuge (Thermo Fisher Scientific, model: Multifuge X1R )
  6. Cell sorter (e.g. FACSaria, BD Biosciences)
  7. 100 µm cell strainer (Corning, catalog numer: 08-771-19 )
  8. Hemocytometer
  9. 15 ml Falcon tubes
  10. 50 ml Falcon tubes
  11. FACS tubes

Procedure

  1. Freshly prepare liver digestion solution by pipetting 4.4 ml Krebs-Ringer-Buffer (KRB), 25 µl DNase I solution, 500 µl Collagenase IV solution, 20 µl 0.5 M CaCl2 and 50 µl 0.2 M MgCl2 into a gentleMACS C tube. Warm up by keeping at 37 °C for 30 min. This solution ensures effective liver tissue digestion while proteolytic degradation of immune cell surface receptors is limited.
  2. Sacrifice mouse by cervical dislocation and remove the liver from a mouse aseptically under the sterile bench and transfer the organ into a well of a sterile 6 well plate filled with 5 ml KRB buffer. If co-isolation of some ILC2 cells potentially present in the vasculature should be avoided include liver perfusion e.g. via the portal vein system using standard techniques described elsewhere.
  3. Rinse the liver with KRB buffer and transfer it into the C tube containing pre-warmed liver digesting solution.
  4. Dissociate the tissue by applying the C tube into the gentleMACS Dissociator and running the program m_liver-01.02.
    Note: One mouse liver can be proceeded per C tube, the program can be run 2-3 times till the tissue is fully homogenized.
  5. Attach the C tube containing the homogenized tissue to the MACSmix Tube Rotator and incubate the sample at 37 °C for 30 min under continuous rotation.
  6. Apply the tube to the gentleMACS Dissociator again and run the program m_liver-02.02.
  7. Prepare a 50 ml tube and a 100 µm cell strainer by rinsing them with PEB buffer.
  8. Apply the homogenized tissue from C tube on the strainer on the 50 ml tube and wash the strainer with additional 10 ml PEB buffer.
  9. Discard the cell strainer and add further 20 ml PEB buffer to the tube. Centrifuge the sample at 20 x g at 4 °C for 4 min. This step removes unwanted hepatocytes from the liver cell suspension.
  10. Carefully remove the supernatant from the sample and transfer it into a new 50 ml tube.
  11. Wash the cells by adding 30 ml PEB buffer and centrifugation at 300 x g for 10 min at 4 °C.
  12. Carefully discard the supernatant and resuspend the cell pellet in 1 ml of PEB buffer.
  13. Apply 10 ml of ACK buffer on the suspension to remove red blood cells. Incubate the sample at room temperature for 5 min.
  14. Wash the sample two times by adding 30 ml PEB buffer and centrifugation at 300 x g for 10 min at 4 °C.
  15. Fill 15 ml tube with 5 ml of 80% Biocoll separation solution.
  16. Resuspend isolated cells in 10 ml 30% Biocoll separation solution and overlay slowly to the 80% Biocoll solution. Centrifuge the cells at room temperature with 1,400 x g for 20 min without breaks.
  17. Collect the target cells by carefully pipetting up the interface fraction between the gradients into a new 50 ml tube.
  18. Wash with 30 ml PEB buffer and centrifuge at 300 x g for 15 min.
  19. Count the cells by using a hemocytometer and distribute to FACS tube for staining.
  20. Prior to staining block unspecific bindings by incubating cells with 1 µl/107 cells of Fc-Block at 4 °C for 5 min.
  21. Proceed immediately to staining using the following antibodies using the manufacturer’s recommended concentrations: anti-Sca-1, anti-ICOS, anti-CD25, biotinylated lineage antibodies. Add 0.015 µg/106 cells of Brilliant Violet 421 Streptavidin.
  22. Incubate the tube at room temperature for 20 min.
  23. Wash the cells by adding 2 ml FACS buffer and centrifuge at 300 x g for 10 min.
  24. Proceed to FACS sorting. Gate and sort ILC2 as lineage negative, Sca-1+, ICOS+, KLRG1+ cells (see gating strategy in Figure 1).


    Figure 1. Gating strategy

Recipes

Note: All materials and reagents should be low endotoxin cell culture quality.

  1. Krebs-Ringer-Buffer (KRB)
    154 mM NaCl
    5.6 mM KCl
    5.5 mM glucose
    20.1 mM NaHCO3
    Adjust pH to 7.4 with NaOH
    Bring to final volume of 1,000 ml
    Filter sterilize and store at 4 °C
  2. Collagenase IV solution
    5,000 Digestion Units/ml in KRB buffer
    Store stock solution at -20 °C
  3. DNase I solution
    30,000 Units/ml in KRB buffer
    Store stock solution at -20 °C
  4. 30% Biocoll
    36.97 ml Biocoll 100%
    63.03 ml PBS
    Keep sterile at 4 °C
  5. 80% Biocoll
    79.8 ml Biocoll 100%
    20.1 ml PBS
    Keep sterile at 4 °C
  6. FACS buffer
    Add 5 ml fetal calf serum to 495 ml of PBS
  7. ACK buffer
    4.145 g NH4Cl
    0.5 g KHCO3
    18.6 g EDTA
    500 ml distilled water
    Filter sterilize and keep at 4 °C
  8. PEB buffer
    Phosphate buffered saline (PBS) containing 0.5 % BSA and 2 mM EDTA

Acknowledgments

This work was supported by the Collaborative Research Center 796 and the Priority program SPP1656 of the DFG (to S.W.) and the Interdisciplinary Center for Clinical Research (IZKF) of the University Medical Center Erlangen.

References

  1. Mchedlidze, T., Waldner, M., Zopf, S., Walker, J., Rankin, A. L., Schuchmann, M., Voehringer, D., McKenzie, A. N., Neurath, M. F., Pflanz, S. and Wirtz, S. (2013). Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis. Immunity 39(2): 357-371.

材料和试剂

  1. 来自IL-33或IL-25处理的小鼠(例如C57BL/6或Balb/c)的肝脏
  2. 胎牛血清(FCS)
  3. RPMI-1640培养基
  4. Easycoll分离溶液(密度1.124)(Biochrom,目录号:L6145)
  5. 抗小鼠CD16/CD32(Fc-Block)(eBioscience,目录号:14-0161)
  6. 0.5M CaCl 2溶液
  7. 0.2M MgCl 2溶液
  8. AutoMACS冲洗溶液(Miltenyi Biotec)
  9. Brilliant Violet 421 Streptavidin(BioLegend,目录号:405226)
  10. 抗体
    1. FACS抗体
      1. 抗Sca-1(克隆D7,FITC缀合的)(eBioscience,目录号:11-5981)
      2. 抗KLRG1(克隆2F1,APC缀合的)(eBioscience,目录号:17-5893)
      3. 抗ICOS(克隆7E.17G9,PE缀合的)(eBioscience,目录号:12-9942)
    2. 抗小鼠谱系抗体生物素标记
      1. 抗CD3(克隆145-2C11)(eBioscience,目录号:13-0031)
      2. 抗CD45R(克隆RA3-6B2)(eBioscience,目录号:13-0452)
      3. 抗Ly-6G(克隆RB6-8C5)(eBioscience,目录号:13-5931)
      4. 抗CD11b(克隆M1/70)(eBioscience,目录号:13-0112)
      5. 抗NK1.1(克隆PK136)(eBioscience,目录号:13-5941)
      6. 抗Ter-119(克隆Ter-119)(eBioscience,目录号:13-5921)
      7. 抗SiglecF(克隆ES22-10D8)(Miltenyi Biotec,目录号:130-101-861)
      8. 抗CD5(克隆53-7.3)(Miltenyi Biotec,目录号:130-101-960)
  11. Krebs-Ringer缓冲液(KRB)(参见配方)
  12. 胶原酶IV溶液(Sigma-Aldrich,目录号:C5138)(参见Recipes)
  13. DNase I溶液(Roche Diagnostics,目录号:10104159001)(参见Recipes)
  14. 30%Biocoll(见配方)
  15. 80%Biocoll(见配方)
  16. FACS缓冲区(参见配方)
  17. ACK缓冲区(参见配方)
  18. PEB缓冲区(参见配方)

设备

  1. GentleMACS C管(Miltenyi Biotec)
  2. 6孔板
  3. GentleMACS解离子(Miltenyi Biotec)
  4. MACSmix管旋转器(Miltenyi Biotec)
  5. 离心机(Thermo Fisher Scientific,型号:Multifuge X1R)
  6. 细胞分选机(例如,FACSaria,BD Biosciences)
  7. 100μm细胞过滤器(Corning,目录号:08-771-19)
  8. 血细胞计数器
  9. 15 ml Falcon管
  10. 50ml Falcon管
  11. FACS管

程序

  1. 通过吸移新鲜制备肝脏消化溶液4.4ml Krebs-Ringer-缓冲液(KRB),25μlDNA酶I溶液,500μl胶原酶IV溶液,20μl0.5M CaCl 2和50μl0.2M MgCl 2 2>进入温和的MACS C管中。 保持在37°C预热30分钟。 这种解决方案确保有效的肝组织消化,而免疫细胞表面受体的蛋白水解降解是有限的。
  2. 通过颈部脱位牺牲小鼠,并在无菌台下无菌地从小鼠中取出肝脏,并将器官转移到填充有5ml KRB缓冲液的无菌6孔板的孔中。 如果共分离 应避免可能存在于脉管系统中的一些ILC2细胞,包括使用其他地方描述的标准技术通过门静脉系统的肝灌注。
  3. 用KRB缓冲液冲洗肝脏,并将其转移到含有预热的肝消化溶液的C管中
  4. 通过将C管应用于温和的MACS解离器并运行程序m_liver-01.02来分离组织。
    注意:每个C管可以进行一个小鼠肝脏,程序可以运行2-3次,直到组织完全均匀化。
  5. 将含有匀浆组织的C管连接到MACSmix管旋转器,并在37℃下连续旋转孵育样品30分钟。
  6. 再次将管子应用到gentleMACS Dissociator并运行程序m_liver-02.02。
  7. 准备一个50毫升管和一个100微米的细胞滤网用PEB缓冲液冲洗
  8. 将取自C管的匀浆组织置于50ml管上的过滤器上,并用另外10ml PEB缓冲液洗涤过滤器。
  9. 弃去细胞过滤器,并向管中再加入20 ml PEB缓冲液。 在4℃下将样品以20×g离心4分钟。 这一步骤从肝细胞悬浮液中去除不需要的肝细胞
  10. 小心地从样品中除去上清液,并将其转移到新的50ml管中
  11. 通过加入30ml PEB缓冲液并在4℃下以300×g离心10分钟来洗涤细胞。
  12. 小心弃去上清液,并将细胞沉淀重悬在1ml PEB缓冲液中。
  13. 应用10毫升ACK缓冲液的悬浮液,以去除红细胞。 在室温下孵育样品5分钟。
  14. 通过加入30ml PEB缓冲液并在4℃下以300×g离心10分钟来洗涤样品两次。
  15. 用5ml 80%Biocoll分离溶液填充15ml管
  16. 将分离的细胞重悬在10ml 30%Biocoll分离溶液中,并缓慢覆盖到80%Biocoll溶液中。 在室温下用1,400×g离心细胞20分钟,不断裂
  17. 通过小心吸取梯度之间的界面部分到新的50ml管中,收集靶细胞
  18. 用30ml PEB缓冲液洗涤并在300×g离心15分钟
  19. 通过使用血细胞计数器计数细胞,并分配到FACS管染色
  20. 在染色之前,通过将细胞与1μl/10μLFc-Block细胞在4℃下孵育5分钟来阻断非特异性结合。
  21. 立即使用以下抗体使用制造商推荐的浓度:抗Sca-1,抗-ICOS,抗-CD25,生物素化谱系抗体进行染色。 加入0.015μg/10 6个细胞的Brilliant Violet 421链霉亲和素。
  22. 在室温下孵育管20分钟。
  23. 通过加入2ml FACS缓冲液洗涤细胞,并在300×g离心10分钟。
  24. 继续FACS分拣。 门控并将ILC2分选为谱系阴性,Sca-1 + ,ICOS + ,KLRG1 + 细胞(参见图1中的门控策略) br />

    图1。 门控策略

食谱

注意:所有材料和试剂应为低内毒素细胞培养质量。

  1. Krebs-Ringer-Buffer(KRB)
    154 mM NaCl 5.6 mM KCl
    5.5mM葡萄糖 20.1mM NaHCO 3/v/v 用NaOH调节pH至7.4 使最终体积为1,000 ml
    过滤灭菌并在4℃下保存
  2. 胶原酶IV溶液
    KRB缓冲液中的5,000消化单位/ml
    将储备液储存在-20°C
  3. DNase I溶液
    在KRB缓冲液
    中为30,000单位/ml 将储备液储存在-20°C
  4. 30%Biocoll
    36.97ml Biocoll 100%
    63.03ml PBS
    保持无菌于4°C
  5. 80%Biocoll
    79.8ml Biocoll 100%
    20.1ml PBS
    保持无菌于4°C
  6. FACS缓冲区
    加入5ml胎牛血清至495ml PBS
  7. ACK缓冲区
    4.145g NH 4 Cl
    0.5g KHCO 3
    18.6g EDTA
    500毫升蒸馏水
    过滤灭菌并保持在4°C
  8. PEB缓冲区
    含有0.5%BSA和2mM EDTA的磷酸盐缓冲盐水(PBS)

致谢

这项工作得到了合作研究中心796和DFG(至S.W.)的优先计划SPP1656和大学医疗中心Erlangen的临床研究跨学科中心(IZKF)的支持。

参考文献

  1. Mchedlidze,T.,Waldner,M.,Zopf,S.,Walker,J.,Rankin,AL,Schuchmann,M.,Voehringer,D.,McKenzie,AN,Neurath,MF,Pflanz,S。和Wirtz,S 。(2013)。 白细胞介素-33依赖性先天淋巴细胞介导肝纤维化。 免疫 39(2):357-371。
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How to cite this protocol: Mchedlidze, T. and Wirtz, S. (2014). Isolation of ILC2 from Mouse Liver. Bio-protocol 4(14): e1179. DOI: 10.21769/BioProtoc.1179; Full Text



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