Biology Reference
In-Depth Information
for 3 min at 37 °C. Inactivate trypsin by adding 2 ml/well of
complete IMDM and pellet the cells by centrifugation;
6. Wash the transduced HEK293T cells once with PBS and pel-
let again by centrifugation;
7. Resuspend the cells in PBS 1× and analyze the percentage of
reporter expressing cells by flow cytometry;
8. The final viral titer is calculated by the following formula,
assuming that the number of HEK293T doubled in the 24 h
between the seeding and the transduction;
+
×
(%
GFPcells
)
ViralTitre
=
×
100 000
,
cells
virusdilutio
nnfactor
100
1. Approximately 12-24 h before transduction, harvest neuro-
spheres by low speed centrifugation (200 ×
g
). Remove the
supernatant and dissociate the neurospheres by resuspending the
pellet in 200 μl of Accutase. Incubate for 10 min at 37 °C and
subsequently dilute the cell suspension in fresh culture medium.
Count the concentration of viable cells using a vital dye;
2. Seed the cells at high density (>2.5 × 10
4
cells/cm
2
) (
see
Note 9
);
3. Before infecting, be sure to have a single cell suspension. Add
the desired amount of virus and gently shake the cell culture
plate/dish/flask. The multiplicity of infection (MOI) is the
ratio between the number of infectious viral particles and the
number of infected cells (
see
Note 10
);
4. Twenty-four hours after transduction, harvest the cells, wash
them once in PBS and plate them again at high density, thus
promoting NPCs recovery after infection and prompt neuro-
sphere reformation (
see
Note 11
);
5. Evaluate the efficiency of transfection at least 7 days after
transduction (
see
Note 12
).
3.3 NPCs
Transduction by
Lentiviral Infection
in Vitro
3.4 Transplantation
of Transduced NPCs
1. At time of transplantation, harvest and dissociate neurospheres
as in Sect.
14.3.1
. NPCs at passage number ≤20 are used in all
experiments. Single cell dissociated NPCs (from 1 to 2 × 10
6
cells in 150 μl PBS) are injected intravenously (i.v.) through
the tail vein. For transplantation on spinal cord the superna-
tant is removed and a volume of ice-cold PBS (without Ca
2+
and Mg
2+
) sufficient for a final concentration of 75 × 10
3
or
150 × 10
3
/μl is added to the pellet and kept in ice prior to
injection.
3.4.1 Preparation
of Cells for Transplant
1. To make vascular access easier, application of heat to the whole
animal or locally to the tail can be used to cause venodilation.
Place the animal into a clear, plastic mouse restraint so that the
animal is not freely mobile, but its tail can be handled;
3.4.2 NPC
Transplantation Through
i.v. Injection
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