Study of Adenovirus and CAR Axonal Transport in Primary Neurons - Adenovirus: Methods and Protocols - page 74

Fig. 1 Hippocampal neurons were incubated with FK for 30 min at 37 °C. After PFA fi xation and permeabiliza-
tion, a specifi c anti-CAR antibody is used to visualize CAR. Arrowheads show colocalization between FK and
endogenous CAR. Scale bars: 5 μ m

5. To monitor FK interaction with CAR, primary neurons can be
incubated with Cy5-FK on ice for 20 min, washed with warm
medium and further incubated at 37 °C for 30 min. Figure 1
shows colocalization between FK and CAR in endosomes.

3.3 Culturing
Motoneurons and
Hippocampal Neurons

1. Primary motoneurons (MNs) ( see Note 3 ) are isolated either
from rat embryonic (E) day 14 or mouse E13 embryos follow-
ing established protocols [ 15 , 16 ]. Briefl y, spinal cords are
removed under a stereomicroscope and trypsined (0.025 %)
for 10 min at 37 °C.
2. Cells are resuspended and dissociated in L15 medium + 0.4 %
BSA + 0.1 % DNAse and centrifuged on a 4 % BSA cushion at
1,000 × g . Cells are resuspended in 1 mL of L15 and selection
for MNs is made by centrifugation (for 15 min at 755 × g at RT
without brake) on a 10.4 % v/v solution of Optiprep ® in L-15.
The interface is then collected in 1 mL of L15, diluted up to
10 mL, and centrifuged on a BSA cushion. Cells are then