Biology Reference
In-Depth Information
based on the property of fibronectin as a permissive substrate for cell pro-
liferation, but not for neuron differentiation in E5 chick neuroretina cell
cultures
(8
,
9)
. This protocol is essentially identical to that described in the
previous section except that cells are always plated on fibronectin and, after
1 h, medium containing [
3
H]methyl thymidine and different ECM molecules
(10
g/mL as final concentration) or laminin-1 proteolytic fragments (in
equimolar amount compared to the native molecule; P1: 3.22
µ
µ
g/mL; E8:
2.66
µ
g/mL) is added.
4. Notes
1.
The time used for the incubation of E5 retinas in the presence of trypsin is crucial
and should be adjusted whenever a new batch of the enzyme is used.
2.
The diameter of the pore of the pipet used for cellular dissociation is crucial for
the quality of the cells and should be empirically tested. In our hands a diameter
of 1-1.5 mm yields the best results.
3.
A control of cell quality should be included. This control is based in the prolifera-
tive capability of the dissociated cells cultured on laminin-1 for 20 h. An increase
of 2.2-2.6-fold in the number of cells should be obtained under these conditions.
Acknowledgments
We thank C. Bauereiss for help with the preparation of
Fig. 1
.
References
1. Adams, J. C. and Watt, F. M. (1993) Regulation of development and differentia-
tion by the extracellular matrix.
Development
117,
1183-1198.
2. Reichardt, L. F. and Tomaselli, K. J. (1991) Extracellular matrix molecules and
their receptors: functions in neural development.
Annu. Rev. Neurosci.
14,
531-570.
3. Timpl, R., Rohde, H., Robey, P. G., Rennard, S. I., Foidart, J. M., and Martin, G. R.
(1979) Laminin - a glycoprotein from basement membranes.
J. Biol. Chem.
254,
9933-9937.
4. Tryggvason, K. (1993) The laminin family.
Curr. Op. Cell Biol.
5,
877-882.
5. Ehrig, K., Leivo, I., Argraves, W. S., Ruoslahti, E., and Engvall, E. (1990)
Merosin, a tissue specific basement membrane protein, is a laminin-like protein.
Proc. Natl. Acad. Sci. USA
87,
3264-3268.
6. Davis, G. E., Manthorpe, M., Engvall, E., and Varon, S. (1985) Isolation and
characterization of rat schwannoma neurite-promoting factor: evidence that the
factor contains laminin.
J. Neurosci.
5,
2662-2671.
7. Prada, C., Puga, J., Pérez-Méndez, L., López, R., and Ramírez, G. (1991) Spatial
and temporal patterns of neurogenesis in the chick retina.
Eur. J. Neurosci.
3,
559-569.
8. Frade, J. M., Martínez-Morales, J. R., and Rodríguez-Tébar, A. (1996a) Laminin-1
selectively stimulates neuron generation from cultured retinal neuroepithelial
cells.
Exp. Cell Res.
222,
140-149.
Search WWH ::
Custom Search