Viral Manipulation of Neural Stem/Precursor Cells - Viral Vector Approaches in Neurobiology and Brain Diseases - page 266

culture dishes (150 × 25 mm). This allows having a sub-
confluent culture (approximately 80 % of confluence) the day
of the transfection;
3. Two hours before transfection, replace the medium with
20 ml of fresh complete IMDM;
4. Prepare the DNA mix by mixing the packaging plasmids and
the transfer vector in a final volume of 1,125 μl of 0.1× TE.
The packaging plasmids commonly used for third generation
lentiviral vectors production are generally coding for gag/pol,
the Rev protein, and the non-correlated envelope of the vesic-
ular stomatitis virus (VSV, protein-G). Usually, the packaging
plasmids are mixed in a 1:1:1 ratio ( see Note 5 );
5. Slowly add 125 μl (i.e., one tenth of the final volume) of 2.5 M
CaCl 2 , mix well, and leave at room temperature for 5 min;
6. While vortexing at maximum speed, add drop wise 1,250 μl of
2× HBS solution to the DNA mix and immediately add the
solution to the culture dish ( see Note 6 );
7. Replace the medium 12-14 h after the transfection, adding
17 ml/dish;
8. After further 30 h, collect the supernatant from the dish and
filter it through 0.22 μm pore-size cellulose acetate filters ( see
Note 7 );
9. Centrifuge the filtered supernatant for 2 h at 100,000 × g at
4 °C;
10. After the centrifugation, discard the supernatant and resuspend
the pellet in an appropriate amount of PBS 1× (e.g., for an
ultracentrifuge tube containing 35 ml of supernatant, resuspend
the pellet in 70 μl so that the lentivirus is concentrated 500×).
To resuspend the pellet, first add the PBS to the bottom of the
ultracentrifuge tube and let it stand on ice for 30 min. Then
gently resuspend the pellet with a p200 pipette and transfer it in
a 1.5 ml tube. Gently shake the tube for 30 min at 4 °C on a
tube rotator to obtain a homogeneous solution ( see Note 8 );
11. Aliquot and store at −80 °C (Fig. 1 ).

Normally, virus titer is expressed as the number of transducing
units (TU) per ml of a production batch. Many lentiviral vectors
usually carry markers/fluorescent reporters, thus allowing func-
tional titration by flow cytometry, and the respective protocol is
described in this section. However, in absence of fluorescent
reporters, titration might be carried out by evaluating the average
number of integrated provirus copies. Viral titration is usually car-
ried out on HEK293T cells but other cell types (including Hela
cell line, HCT116 cell line and NPCs) can be used as well.

3.2.2 Lentivirus Titration