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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

JL Julia Ladewig
PK Philipp Koch
Oliver Brüstle Oliver Brüstle

发布: 2015年01月05日第5卷第1期 DOI: 10.21769/BioProtoc.1371 浏览次数: 19028

评审: Xuecai GeAnonymous reviewer(s)

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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

  1. 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)
  2. DMEM/F12 (high glucose) (Life Technologies, catalog number: 11320074 )
  3. Neurobasal medium (Life Technologies, catalog number: 21103049 )
  4. B27 supplement (Life Technologies, catalog number: 17504044 )
  5. N2 supplement (GE Healthcare, catalog number: T1129,2205 )
  6. Glucose (Roche Diagnostics, catalog number: HN06.3 )
  7. Trypsin/EDTA (Life Technologies, catalog number: 15400054 )
  8. Trypsin inhibitor (Life Technologies, catalog number: 17075029 )
  9. DNAse (CellSystems Biotechnologie Vertrieb GmbH, catalog number: LS002140 )
  10. Specific for matrigel migration assay
    1. MatrigelTM Basment Membrane Matrix (BD Bioscience, catalog number: 354230 ) or equivalent such as Geltrex® Matrix (Life Technologies, catalog number: A1413202 )
    2. 4 well tissue culture dish (VWR International, catalog number: 176740 )
    3. Rock inhibitor (Cell guidance systems, catalog number: SM02-100 )
  11. Specific for Boyden chamber migration assay
    1. Millicell culture plate inserts 8-μm pore size (Millipore, catalog number: PI8P01250 )
    2. Nylon mesh (Pall, http://www.pall.com)
    3. 24 well plate (VWR International, catalog number: 734-0056 )
    4. Poly-l-ornithine (Sigma-Aldrich, catalog number: P3655-1G )
    5. Laminin (Life Technologies, catalog number: 23017015 )
    6. PBS (Life Technologies, catalog number: 14190094 )
    7. Chemoattractant such as FGF2 (R&D Systems, catalog number: 233-FB/CF ) or VEGF (R&D Systems, catalog number: 236-EG-01M )
    8. 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)
    9. Cotton bud (drug store, e.g. Q-tip®)
    10. Solutions and antibodies for standard immunohistochemical staining
  12. Neural stem cell media (see Recipes)
  13. Neuronal differentiation media (see Recipes)

Equipment

  1. Cell culture centrifuge (e.g., Megafuge 1.0 R, Kendro Laboratory)
  2. 37 °C, 5% CO2 cell culture incubator (e.g., Thermo Fisher Scientific, model: Heracell 240 )
  3. Counting chamber (e.g., Fuchs-Rosenthal, Marienfeld)
  4. Glass object slide (e.g., Thermo Fisher Scientific, Superfrost Plus)
  5. Microscope (e.g., ZEISS, model: Axiovert 200M )

Procedure

English
中文翻译

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.

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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

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