In vitro Migration Assays for Neural Stem Cells, Intermediate Neurogenic Progenitors and Immature Neurons
神经干细胞、中间神经性前体细胞和未成熟神经元的体外迁移分析
发布: 2015年01月05日第5卷第1期 DOI: 10.21769/BioProtoc.1371 浏览次数: 18530
评审: Xuecai GeAnonymous reviewer(s)
PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Jan 2014
Abstract
In the vertebrate central nervous system (CNS), different neural precursor populations such as neural stem cells (NSCs), intermediate neurogenic progenitors (INPs) and immature neurons have to migrate from their places of birth to their location of function. Coordinated migration is mediated by direct cell-cell interactions and by extracellular matrix components, chemoattractants as well as repellents. The migration potential of such populations as well as the responsiveness to chemoattractive compounds can be addressed in isolated cells using in vitro migration assays. Here we describe two migration assays, a matrigel migration assay and a Boyden chamber migration assay, which allow the in vitro assessment of neural migration under defined conditions (Ladewig, Koch and Brüstle, 2014). A matrigel matrix is a soluble basement membrane extract. The major components of matrigel matrix are collagens, laminin and proteoglycans, which provide the substrate for migrating cells. In the matrigel assay migration can be analyzed using a phase contrast microscope. The Boyden chamber assay (Richards and McCullough, 1984) is based on microchemotaxis chambers, which consist of two compartments separated by a membrane with a defined pore size. Cells can be plated in the upper compartment and allowed to migrate through the pores towards the lower compartment, in which a potential chemotactic agent is loaded. Cell migration can be analyzed following fixing and immunohistochemical staining. In principle, the described protocols should be applicable to other cell populations such as endothelial cells or cancer cells using conditions adapted to the individual needs of the specific cell type.
Materials and Reagents
- Neural cell populations: e.g. pluripotent stem cell (PSC) derived neuroepithelial stem cells (lt-NES) and neurons derived thereof (Ladewig et al., 2008; Koch et al., 2009)
- DMEM/F12 (high glucose) (Life Technologies, catalog number: 11320074 )
- Neurobasal medium (Life Technologies, catalog number: 21103049 )
- B27 supplement (Life Technologies, catalog number: 17504044 )
- N2 supplement (GE Healthcare, catalog number: T1129,2205 )
- Glucose (Roche Diagnostics, catalog number: HN06.3 )
- Trypsin/EDTA (Life Technologies, catalog number: 15400054 )
- Trypsin inhibitor (Life Technologies, catalog number: 17075029 )
- DNAse (CellSystems Biotechnologie Vertrieb GmbH, catalog number: LS002140 )
- Specific for matrigel migration assay
- Specific for Boyden chamber migration assay
- Millicell culture plate inserts 8-μm pore size (Millipore, catalog number: PI8P01250 )
- Nylon mesh (Pall, http://www.pall.com)
- 24 well plate (VWR International, catalog number: 734-0056 )
- Poly-l-ornithine (Sigma-Aldrich, catalog number: P3655-1G )
- Laminin (Life Technologies, catalog number: 23017015 )
- PBS (Life Technologies, catalog number: 14190094 )
- Chemoattractant such as FGF2 (R&D Systems, catalog number: 233-FB/CF ) or VEGF (R&D Systems, catalog number: 236-EG-01M )
- Potential inhibitors for used chem¬oattractant such as VEGF receptor 1 and VEGF receptor 2 blocking antibodies (both R&D Systems, catalog numbers: AF321 and AF357 ) and the FGF2 neutralizing antibody (Millipore, clone bFM-1)
- Cotton bud (drug store, e.g. Q-tip®)
- Solutions and antibodies for standard immunohistochemical staining
- Millicell culture plate inserts 8-μm pore size (Millipore, catalog number: PI8P01250 )
- Neural stem cell media (see Recipes)
- Neuronal differentiation media (see Recipes)
Equipment
- Cell culture centrifuge (e.g., Megafuge 1.0 R, Kendro Laboratory)
- 37 °C, 5% CO2 cell culture incubator (e.g., Thermo Fisher Scientific, model: Heracell 240 )
- Counting chamber (e.g., Fuchs-Rosenthal, Marienfeld)
- Glass object slide (e.g., Thermo Fisher Scientific, Superfrost Plus)
- Microscope (e.g., ZEISS, model: Axiovert 200M )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Ladewig, J., Koch, P. and Brüstle, O. (2015). In vitro Migration Assays for Neural Stem Cells, Intermediate Neurogenic Progenitors and Immature Neurons. Bio-protocol 5(1): e1371. DOI: 10.21769/BioProtoc.1371.
Download Citation in RIS Format
分类
神经科学 > 细胞机理 > 细胞分离和培养
发育生物学 > 细胞生长和命运决定 > 神经元
细胞生物学 > 细胞运动 > 细胞迁移
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
