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
Equipment
Procedure
Representative data
Figure 1. (A-B) Matrigel migration assay addressing the migration capacity of lt-NES cell derived purified neurons in comparison to neurons within non-purified population. Equal numbers of cells were plated on matrigel matrix and analyzed 48 h later. Purified neurons showed a radial symmetric distribution across a large area with migration of individual neurons from the plating site A. In contrast, the non-purified population (containing neurons as well as progenitor cells) formed spherical clusters with hardly any neurons leaving these aggregates. Instead, radial axonal outgrowth was observed B. Scale bar: A-B. 200 µm A B Figure 2. Cell migration studied by using millicell culture plate inserts. A. Cells are plated on the membrane of the upper well. Chemoattractants can be added to the lower well. Migration of cells from the upper well through the membrane can be measured by wiping off the remaining cells from the upper side of the membrane using a cotton bud and counting the cells that reached the bottom side. Adapted from Erlandsson (2003). B. Assessing the migration of neurons within a cell mixture (containing neurons and progenitor cells) and of purified neurons. Bars represent the percentage of neurons reaching the lower side of the membrane after 20 h. Cell numbers were normalized to the number of neurons plated and shown as mean+SD (*P < 0.05).
Notes
The quality of the Millicell culture plate inserts can vary, thus it is crucial to ensure that the cell destribution is uniform on both the upper and lower side of the membrane as described in point 11 of the Boyden chamber migration protocol.
Recipes
Acknowledgments
This work was supported by the European Union (grant 222943 Neurostemcell; LSHG-CT-2006-018739, ESTOOLS), the German Research Foundation (DFG; SFB-TR3), the Hertie Foundation and the Ministry of Innovation Science and Research of North Rhine-Westphalia (Junior Research Group, L-072.0081).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.