Biology Reference
In-Depth Information
plated in culturing medium on coated glass-bottom dishes
(90 min with 15
μ
g/mL poly-ornithine and overnight with
g/mL of laminin). All imaging experiments are performed
using neurons differentiated in vitro for 5-8 days.
3. Primary hippocampal neurons are prepared from E18 mice
fetuses. Embryos are taken out and immediately transferred in
dissecting media (PBS-Glucose 3 %). Hippocampi (banana-
shaped) are microdissected under the stereomicroscope after
separation of the two cortical lobes and meninges.
4. Hippocampi are trypsined (0.025 %) for 5 min at 37 °C.
Hippocampal cells are then dissociated in 2 mL L15
medium + 0.4 % BSA + 0.1 % DNAse. Cell suspension is centri-
fuged for 5 min at 1,000 ×
g
and the pellet is resuspended in
Neurobasal containing B27,
L
-glutamine, Glutamax, 10 % fetal
calf serum, and antibiotics. Cells are then plated on coated
glass-bottom dishes (90 min with 15
3
μ
g/mL poly-ornithine).
All imaging experiments are performed using neurons differ-
entiated in vitro for 7-10 days.
μ
1. MNs are incubated with fl uorescent ligands (2,000 pp/cells of
CAdV2-Cy3 or 1.5
3.4 Live-Cell
Imaging and Analyses
of CAdV-2 and FK
Axonal Transport
g/mL of Cy5-FK) for 30-60 min, then
washed with warm HBSS (Hanks Balanced Salt Solution)-
HEPES pH 7.4. Cells are then imaged by confocal microscopy
(e.g., Zeiss LSM 510 or 780 equipped with a 63× 1.4 NA Plan
Apochromat oil-immersion objective).
2. After axons are identifi ed morphologically (thinnest process),
100-150 frames are acquired at a rate of 0.4 frame/s (
see
Note 4
).
Images and movies are processed using the LSM 510 or Zen
software (Fig.
2a
). Kymographs can be generated using
MetaMorph (Molecular Devices) by tracing a single line
encompassing axons and plotting sequentially every frame of
the movie (Fig.
2b
).
3. Alternatively, to study axonal transport, microfl uidic chambers
(MCs, compartmentalized chambers), can be used to isolate
fl uidically axons from the cell body. In these devices, axons can
grow through a parallel array of microchannels connecting two
compartments (Fig.
2c, d
). MCs are coated with 4 % BSA for
2 h, then 15
μ
g/
mL laminin for 1 h 30. All these incubations are performed at
37 °C and three PBS washes are needed between poly-orni-
thine and laminin. 150,000 hippocampal neurons per MC are
then plated in one compartment. To force axonal growth into
the microgrooves, BDNF (20 ng/mL fi nal) is added in the
axonal compartment after 4 days. 8-10 days after cell plating,
fl uorescent ligands can be added to the axonal compartment in
150
μ
g/mL poly-ornithine for 2 h, and with 50
μ
μ
L of culturing medium. The cell body compartment
