Biology Reference
In-Depth Information
Soybean trypsin inhibitor (Sigma) (10 mg/mL in sterile Ca 2+ -, Mg 2+ -free PBS).
Aliquots may be stored at -20
5.
°
C.
6.
DMEM/F12 Ham (1:1) medium (Sigma) with N2 supplements (see below) . 100 mL
medium should contain 10 mg transferrin (Sigma), 1 mL insulin solution (Sigma)
(0.5 mg/mL in sterile Ca 2+ -, Mg 2+ -free PBS containing 4
µ
L/mL concentrated
HCl, this solution may be stored at 4
L putrescin solution (Sigma)
(160 mg/mL in sterile Ca 2+ -, Mg 2+ -free PBS), 10
°
C), 10
µ
µ
L progesterone solution
(Sigma) (60
µ
g/mL in 70%), 10
µ
L sodium selenite solution (Sigma) (52
µ
g/mL in
sterile Ca 2+ -, Mg 2+ -free), 100
g/mL; this
solution is supplied by the manufacturer ready to use and may be stored at 4
µ
L gentamicin solution (Sigma) (50
µ
°
C;
all other solutions, unless otherwise indicated can be stored at -20
C for more
than 1 yr). Ensure that the pH of the medium is in the range of 7.15-7.25.
°
7.
[ 3 H]methyl thymidine (25 Ci/mmol) (Amersham, Arlington Heights, IL).
8.
G4 monoclonal antibody (12) , ascitic fluid.
9.
Krebs-Ringer-HEPES (KRH): 125 m M NaCl (7.30 g/L), 4.8 m M KCl (0.36 g/L),
1.3 m M CaCl 2 · 2H 2 O (0.19 g/L), 25 m M free-acid HEPES (5.96 g/L), 1.2 m M
MgSO 4 · 7H 2 O (0.30 g/L), 1.2 m M KH 2 PO 4 (0.16 g/L), 5.6 m M Glucose (1.11 g/L);
pH 7.3. All reagents were from Merck, except HEPES, that came from Sigma.
10.
Biotinylated sheep antimouse IgG species-specific whole antibody (Amersham).
11.
Streptavidin-texas red (Amersham).
4% paraformaldehyde solution in Ca 2+ -, Mg 2+ -free PBS (heat 100 mL of this
buffer containing 4 g paraformaldehyde and 130
12.
L 1 M NaOH until the solution
becomes transparent, this solution should be freshly made).
µ
13.
DePeX (Serva, Heidelberg, Germany).
14.
NTB-2 photographic emulsion (Kodak)/2% glycerol in water (1:2). In the dark-
ness, add one volume of solid NTB-2 emulsion to two volumes of 2% glycerol
prewarmed to 42
°
C. Incubate at 42
°
C for 15 min and mix the solution avoiding
the production of air bubbles.
15. D19 developer (Kodak) diluted 1:1 in water.
16. 25% sodium thiosulfate (Na 2 S 2 O 3 ) (Sigma) in water.
17. Ca 2+ -, Mg 2+ -free PBS/Glycerol(1:1).
3. Methods
E5 chick retina consists mostly of neuroepithelial cells in active prolifera-
tion. These cells can be easily dissociated to obtain single cells (see below) and
thus the influence of particular ECM molecules in neuron differentiation can
be analyzed in vitro. After 20 h in culture, cells labeled with [ 3 H]methyl thymi-
dine and a neuronal marker represent neuroepithelial cells that were still in the
process of cell division at the time of plating and that have acquired neuronal
properties during the time in culture (pipet in vitro differentiated neurons).
Here we will describe how to quantify neuronal differentiation using as neu-
ronal marker the glycoprotein G4. This molecule is a member of the immuno-
globulin superfamily of cell adhesion molecules that is expressed mostly by
retinal ganglion cells but not neuroepithelial cells (12) .
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