Isolation and Purification of Murine Microglial Cells for Flow Cytometry
分离和纯化鼠小胶质细胞并进行流式细胞术
发布: 2016年01月05日第6卷第1期 DOI: 10.21769/BioProtoc.1703 浏览次数: 15379
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参见作者原研究论文
The authors used this protocol in:
Apr 2015
Abstract
The detailed protocol is used to isolate different cell types from murine brain as glial cells, including microglia, using an enzymatic digestion that minimizes cellular mortality. A Percoll gradient (30% to 80%) separation allows a maximal recovery of isolated murine microglial cells prior to flow cytometry analysis.
Keywords: Flow Cytometry (流式细胞仪)
Microglial cell (小胶质细胞)
Mouse (小鼠)
Brain (脑)
Materials and Reagents
- Syringe 10 ml (BD bioscience, catalog number: 309604 )
- Needle 22 G (BD bioscience, catalog number: 305155 )
- Syringe 1 ml 27 G (BD bioscience, catalog number: 309623 )
- 15 ml centrifugation tube (Thermo Fisher Scientific, catalog number: 339651 )
- 50 ml centrifugation tube (Thermo Fisher Scientific, catalog number: 339653 )
- 96 v-shaped wells plate (Thermo Fisher Scientific, catalog number: 249662 )
- 5 ml polystyrene FACS tube (BD Biosciences, Falcon®, catalog number: 352054 )
- 70 µm nylon filter (VWR International, catalog number: 352350 )
- Mice
- PBS without Ca2+/Mg2+ (BOLLÉ COMMUNICATIONS, Wisent, catalog number: 311-010-CL )
- Ice cold Dulbecco’s Phosphate Buffered Saline (DPBS) without Ca2+/Mg2+/ Dulbecco’s Phosphate Buffered Saline (Sigma-Aldrich, catalog number: D8537 )
- PBS 10x without Ca2+/Mg2+ (BOLLÉ COMMUNICATIONS, Wisent, catalog number: 311-012-CL )
- Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) (Sigma-Aldrich, catalog number: T7254 )
- Liberase TL Research Grade (Roche Diagnostics, catalog number: 05401020001 )
- DNAse1 (Roche Diagnostics, catalog number: 11284932001 )
- HEPES (1 M) (Sigma-Aldrich, catalog number: H0887 )
- Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F1051 )
- EDTA (Sigma-Aldrich, catalog number: E16144 )
- Percoll (GE Healthcare, Dharmacon, catalog number: 17-0891-01 )
- HBSS 10x without Ca2+/Mg2+ (BOLLÉ COMMUNICATIONS, Wisent, catalog number: 311-506-CL )
- HBSS without Ca2+/Mg2+ with red phenol (Life technologies, catalog number: 14170-112 )
Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 14170-112”.
- Purified Rat anti-mouse CD16/CD32 (BD Bioscience, catalog number: 553141 )
- Anti-mouse CD45 PE/Cy5 (BD Bioscience, catalog number: 553082 )
- Anti-mouse CD11b Alexa 700 (eBioscience, catalog number: 56-0112-82 )
- Anti-mouse CD16/CD32 (1:100) (BD Biosciences, catalog number: 553142 )
- Live/Dead Fixable Blue Dead Cell (Life technologies, catalog number: L23105 )
Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: L23105”.
- Digestion medium (see Recipes)
- Ice-cold FACS buffer (see Recipes)
- Percoll dilution medium (see Recipes)
- PFA 4% (w/v) solution (see Recipes)
Equipment
- Tissue grinder Capacity 3 ml (VWR International, catalog number: 886000-0020 )
- Centrifuge Sorvall legend RT (Mandel, catalog number: 75004377 )
- Flow cytometer BD Canto II or LSR II (BD bioscience)
- Peristaltic pump, facultative (Cole-Parmer Instrument Company, catalog number: EW-78001-02 )
Software
- BD FACS Diva software (version 6.1.2)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Theriault, P., Bordeleau, M. and Rivest, S. (2016). Isolation and Purification of Murine Microglial Cells for Flow Cytometry. Bio-protocol 6(1): e1703. DOI: 10.21769/BioProtoc.1703.
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分类
神经科学 > 细胞机理 > 细胞分离和培养
细胞生物学 > 基于细胞的分析方法 > 流式细胞术
细胞生物学 > 细胞分离和培养 > 细胞分离
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