Isolation and FACS Analysis on Mononuclear Cells from CNS Tissue
从CNS组织分离单核细胞及FACS分析
发布: 2014年09月20日第4卷第18期 DOI: 10.21769/BioProtoc.1240 浏览次数: 13778
评审: Kanika GeraAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2013
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Abstract
Immune cells, such as microglia are resident in the brain and spinal cord of normal mice and humans. Furthermore, macrophages, dendritic cells, T cells, B cells and NK cells infiltrate the CNS during certain infections or in neurodegenerative/neuroinflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE) (a model for multiple sclerosis) or Alzheimer’s disease (Sutton et al., 2009; Browne et al., 2013). Infiltrating cells can be identified using immunohistological staining of sections from brain or spinal cords. However, more detailed phenotypic and functional analysis is possible following isolation of the immune cells from the CNS of normal or diseased mice. Purification of mononuclear cells from brain or spinal cord is dependent on perfusing the mouse to ensure removal of the blood from the CNS tissue, prior to dissociating the tissue and purification of the mononuclear cells on a percoll gradient. The technique provides single cell suspensions with cells of high viability that are suitable for FACS analysis or limited functional studies. The yields are usually low from the normal mouse brain or spinal cord, but higher from mice with EAE or CNS infection. When combined with intracellular cytokine staining and FACS, this technique is particularly useful for analysis of the pathogenic T cells (Th17 and Th1 cells) and their regulation/modulation in EAE.
Materials and Reagents
- Mice (adult >6 weeks, any strain, e.g. C57BL/6 used for MOG-induced EAE)
- Sodium pentobarbital (euthatal) (Merial)
- Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537 )
- 10x PBS (Sigma-Aldrich, catalog number: D1408 )
- RPMI solution(Sigma-Aldrich, catalog number: R0883 )
- Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333 )
- L-glutamine (Sigma-Aldrich, catalog number: G7513 )
- FBS (Sigma-Aldrich, catalog number: F9665 )
- Hank’s balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H9394 ) supplemented with 3% FBS
- Collagenase D (Roche, catalog number: 11088858001 )
- DNase I (Sigma-Aldrich, catalog number: D4263 )
- Percoll Plus (Sigma-Aldrich, catalog number: 17-5445-01 )
- Cell permeabilisation kit (contains IntraStain Reagent A and B) (Dako, Denmark, catalog number: K2311 )
- Phorbol myristate acetate (PMA) (Sigma-Aldrich, catalog number: P1585 )
- Ionomycin (Sigma-Aldrich, catalog number: I0634 )
- Brefeldin A (BFA) (Sigma-Aldrich, catalog number: B7651 )
- LIVE/DEAD® Fixable Aqua Dead Cell Stain kit (Life Technologies, catalog number: L34957 )
- CD16/CD32 FcγRIII (BD Biosciences, catalog number: 553141 )
- FACS antibodies (as appropriate)
- Propidium iodide (PI) (Sigma-Aldrich, catalog number: P4864 )
- Complete RPMI solution (see Recipes)
- FACS buffer (see Recipes)
- Stock isotonic percoll (SIP) (see Recipes).
Equipment
- 70 μm nylon mesh filter (Corning, catalog number: 352350 )
- Shaker capable of 200 rpm at 37 °C
- Tissue culture facilities including class II laminar flow hood
- Bench-top centrifuge, preferably refrigerated (any model)
- Flow cytometer
Software
- Summit software (Dako)
- FlowJo software (Tree Star)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Mills, K. H., McManus, R. M. and Dungan, L. (2014). Isolation and FACS Analysis on Mononuclear Cells from CNS Tissue. Bio-protocol 4(18): e1240. DOI: 10.21769/BioProtoc.1240.
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分类
免疫学 > 免疫细胞染色 > 流式细胞术
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
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