Isolation of ILC2 from Mouse Liver
从小鼠肝脏中分离第2组先天淋巴细胞（ILC2）
发布: 2014年07月20日第4卷第14期 DOI: 10.21769/BioProtoc.1179 浏览次数: 13281
评审: Savita NairAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2013
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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)
Materials and Reagents
- Liver from a IL-33 or IL-25 treated mouse (e.g. C57BL/6 or Balb/c)
- Fetal calf serum (FCS)
- RPMI-1640 medium
- Easycoll separation solution (density 1.124) (Biochrom, catalog number: L6145 )
- Anti-mouse CD16/CD32 (Fc-Block) (eBioscience, catalog number: 14-0161 )
- 0.5 M CaCl2 solution
- 0.2 M MgCl2 solution
- AutoMACS rinsing solution (Miltenyi Biotec)
- Brilliant Violet 421 Streptavidin (BioLegend, catalog number: 405226 )
- Antibodies
- FACS antibodies
- Anti-mouse lineage-antibodies biotin labelled
- Anti-CD3 (clone 145-2C11) (eBioscience, catalog number: 13-0031 )
- Anti-CD45R (clone RA3-6B2) (eBioscience, catalog number: 13-0452 )
- Anti-Ly-6G (clone RB6-8C5) (eBioscience, catalog number: 13-5931 )
- Anti-CD11b (clone M1/70) (eBioscience, catalog number: 13-0112 )
- Anti-NK1.1 (clone PK136) (eBioscience, catalog number: 13-5941 )
- Anti-Ter-119 (clone Ter-119) (eBioscience, catalog number: 13-5921 )
- Anti-SiglecF (clone ES22-10D8) (Miltenyi Biotec, catalog number: 130-101-861 )
- Anti-CD5 (clone 53-7.3) (Miltenyi Biotec, catalog number: 130-101-960 )
- Anti-CD3 (clone 145-2C11) (eBioscience, catalog number: 13-0031 )
- Krebs-Ringer-Buffer (KRB) (see Recipes)
- Collagenase IV solution (Sigma-Aldrich, catalog number: C5138 ) (see Recipes)
- DNase I solution (Roche Diagnostics, catalog number: 10104159001 ) (see Recipes)
- 30% Biocoll (see Recipes)
- 80% Biocoll (see Recipes)
- FACS buffer (see Recipes)
- ACK buffer (see Recipes)
- PEB buffer (see Recipes)
Equipment
- GentleMACS C tube (Miltenyi Biotec)
- 6 well plates
- GentleMACS dissociator (Miltenyi Biotec)
- MACSmix tube rotator (Miltenyi Biotec)
- Centrifuge (Thermo Fisher Scientific, model: Multifuge X1R )
- Cell sorter (e.g. FACSaria, BD Biosciences)
- 100 µm cell strainer (Corning, catalog numer: 08-771-19 )
- Hemocytometer
- 15 ml Falcon tubes
- 50 ml Falcon tubes
- FACS tubes
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Mchedlidze, T. and Wirtz, S. (2014). Isolation of ILC2 from Mouse Liver. Bio-protocol 4(14): e1179. DOI: 10.21769/BioProtoc.1179.
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分类
免疫学 > 免疫细胞分离 > 淋巴细胞
细胞生物学 > 细胞分离和培养 > 细胞分离
细胞生物学 > 组织分析 > 组织分离
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