Biology Reference
In-Depth Information
1. Dulbecco's Modifi ed Eagle's Medium supplemented with 10 %
fetal calf serum and antibiotics (100 g/L Streptomycine/10
8
U/L
Penicilline; diluted 2,500 times). For DKZeo culture, nones-
sential amino acids (NEAA) and G418 (fi nal concentration,
0.5 mg/mL) are added.
2.2 Cell Culture
2. 0.25 % Trypsin.
3. Turbofect reagent.
4. 10× Hanks Balanced Salt Solution diluted in water and adjusted
to pH 7.4 with HEPES.
5. Polyornitine dissolved in sterile water at 1.5 mg/mL, fi ltered,
and stored aliquoted at −20 °C.
6. 1 mg/mL Laminin.
7. Glucose.
8. Leibovitz's L-15 medium.
9. Bovine serum albumine.
10. Deoxyribonuclease I (DNAse) resuspended in Leibovitz's
L-15 medium at 20 mg/mL, fi ltered and stored aliquoted at
−20 °C.
11. 50× B27 Supplement.
12.
L
-Glutamine (200 mM—use at 2 mM).
13. 100× Glutamax.
14. Neurobasal medium.
15. Optiprep
®
.
16. Culture medium for motor neurons and hippocampal neurons:
Neurobasal, Glutamax, B27, 0.5 % horse serum, 10 ng/mL
CTNF, 200 pg/mL GNDF, and antibiotics.
1. Glass-bottom dishes.
2. Microfl uidic chambers can be made accordingly to published
methods [
11
] or bougth at Millipore (AXIS™ Axon Isolation
Devices) and covalently bound to glass-bottom dishes [
11
,
12
].
2.3 Confocal
Microscopy
3
Methods
1.
Δ
E1-deleted CAdV-2 vectors are produced and purifi ed fol-
lowing established protocol [
13
]. Briefl y, 12
3.1 Production
and Labeling of
CAdV-2 Vectors
μ
g of linearized
L of turbofect in 3
wells of a 6-well plate of DKZeo cells cultivated in DMEM
(supplemented with FCS, NEAA, and G418) for 5 days.
2. Cells are then scraped and go through three cycles of freeze/
thaw. After centrifugation at 5,000 ×
g
at RT for 10 min to
eliminate debris, supernatant is applied to a 10 cm Ø dish of
pCAV-Cre plasmid are transfected with 24
μ
