Neuroepithelial Differentiation Induced by ECM Molecules - Methods in Molecular Biology: Extracellular Matrix Protocols - page 246

simple compared with other regions within the CNS, it is completely isolated
from other CNS structures, a feature that simplifies its dissection, and it is
completely avascular, therefore, there is no contamination from nonnervous
tissues during the process of dissection. The embryonic day 5 (E5) chick retina
consists of a neuroepithelium mainly composed of precursor cells, which are
undergoing active neurogenesis (7) . This structure has been the object of active
study and conditions that permit neurogenesis in cultures of dissociated neu-
roepithelial cells are well known (8 , 9) . Among these conditions, the presence
of at least the long arm of laminin-1 is absolutely required (8) .
Here we will describe a set of protocols that can be used for the analysis of
neuronal differentiation induced by ECM molecules using dissociated E5 chick
retina neuroepithelial cells in culture.

2. Materials
2.1. Coating with ECM Substrata
1. 10 mm round, coverslips (Menzel-Gläser, Germany).

2. 65% nitric acid.

3. Sterile distilled water.

4. Absolute ethanol.

5. 35/10-mm four-well Petri dishes (Greiner, Fickenhausen, Germany).

6. Poly (D-L) ornithine (Cat. no. P8638) (Sigma, St. Louis, MO) (0.5 mg/mL in

150 m M Na borate, pH 8.4). Aliquots may be stored at -20

° C for more than 1 yr.

Ca 2+ -, Mg 2+ -free PBS: 136.89 m M NaCl (8 g/L), 2.68 m M KCl (0.2 g/L), 6.46 m M
Na 2 HPO 4 . 2H 2 O (1.15 g/L), 1.47 m M KH 2 PO 4 (0.2 g/L), pH 7.3 (autoclaved). All
reagents from Merck (Darmstadt, RFA).

7.

8. Laminin-1 (10 g/mL, final concentration, in Ca 2+ , Mg 2+ -free PBS) (Gibco-BRL,

Gaithersburg, MD, or Collaborative Biomedical Products, New Bedford, MA).

µ

9. Merosin (10 µ g/mL; final concentration) (Gibco-BRL).

10. Schwannoma laminin (10 µ g/mL; final concentration) (6) .

11. Fibronectin (10 µ g/mL; final concentration) (Gibco-BRL).

12. Laminin-1 proteolytic fragments P1 and E8 (10) . These fragments are used in

soluble form, they are added in equimolar amount as that of laminin-1 to cells
plated onto fibronectin (P1: 3.22 µ g/mL; E8: 2.66 µ g/mL; final concentration in

Ca 2+ , Mg 2+ -free PBS).

2.2. The Differentiation Bioassay
1. Chick embryos staged as described elsewhere (11) .

2. Material for dissection (dissecting scissors, Dumont #5 tweezers, and curved

iris forceps).

Bovine serum albumin (BSA) (Sigma) (30 mg/mL in sterile Ca 2+ -, Mg 2+ -free
PBS). Aliquots may be stored at -20

3.

° C for more than 1 year.

4. Trypsin (Worthington). The powder should be aliquoted and stored at 4 C

under dry conditions for less than 1 year (storage conditions are very impor-
tant for reproducibility)

°