Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 200

The relative titer/ml is calculated by applying the following
formula:
(number of RNA copies × dilution of vector preparation)/
volume in ml.

293T and HeLa cells are used for titer determination by fl ow
cytometry (fl uorescence-activated cell sorting, FACS), but this
approach is suitable only for LVs expressing reporter genes encod-
ing fl uorescent proteins (GFP, for example) under the control of a
promoter active in these cells. Each well of a 24-well plate are
seeded with 1 × 10 5 293T cells. On the next day, the cells are
infected with serial dilutions of LVs, from 1/10 to 1/10 7 , pre-
pared in DMEM. Either 48 h or 72 h (for LV-MOK-G) after trans-
duction, the cells are washed with PBS and dissociated by treatment
with 150

3.7.3 Determination
of the Function Titer
by Flow Cytometry

l) is then added to inac-
tivate the trypsin. The cells are then fi xed by incubation with 300

μ l of 1× Trypsin. DMEM (150 μ

l
of 2 % formaldehyde in PBS and transferred to FACS tubes ( see
Note 17 ). Flow cytometry analysis is performed to determine the
percentage of cells positive for GFP ( see Note 18 ).
Titer is calculated as follows:
% of positive cells × number of cells on the day of infec-
tion × dilution factor ( see Note 19 ).

μ

Primary cultures are set up in a laminar fl ow biohazard hood in a
BSL2 laboratory. The adhesion of the cells to plastic is enhanced by
coating the multiwell plates with 5 % poly- D -lysine (400

3.8 Culture and
Transduction of
Primary Striatal
Neurons

l/well or
1.5 ml/well for a 24- or a 6-well plate, respectively) the day before
culture and incubating the plates overnight in a humidifi ed incuba-
tor at 37 °C under an atmosphere containing 5 % CO 2 . The next
day, the wells are rinsed with sterile water and dried under the hood.

μ

Cocultures of primary striatal neurons are prepared from E15 rats
(Sprague Dawley rats). A pregnant rat is anesthetized fi rst with
isofl urane and then with pentobarbital (injection below the fi rst
breast). The skin is disinfected with alcohol, the abdominal cavity
opened, and the uterine horns are withdrawn. Embryos are placed
in HBSSm (see Sect. 2 for composition) and their heads are cut off.
From this point onwards, all the material must be kept on ice.
Brains are retrieved under a dissecting microscope ( see Note 20 )
and placed on a large Petri dish containing HBSSm. The striata are
dissected out ( see Note 21 ) and collected in 5 ml of HBSSm in a
Petri dish. They are then thinly sliced with microscissors. HBSSm
(4 ml) and 1 ml of DNase I (3 mg/ml) are added and the mixture
is incubated for 15 min at 37 °C. The cells are vigorously dissoci-
ated with a 1 ml tip, to give a turbid suspension ( see Note 22 ). The
mixture is allowed to settle for 3 min on ice and 9 ml is collected
in a second tube. HBSSm (1 ml) is then added to the rest of the
mixture for complete dissociation of the cells. This mixture is

3.8.1 Cocultures
of Primary Striatal Neurons