Viral Manipulation of Neural Stem/Precursor Cells - Viral Vector Approaches in Neurobiology and Brain Diseases - page 267

Fig. 1 Schematic workflow of the production of the viral particles. The viral vector
with the insert of interest is transfected along with the plasmids for the packag-
ing system in the 293 T cell line. The supernatant with the viral particles is col-
lected and concentrated by ultracentrifugation
1. Seed 5 × 10 4 HEK293T cells per well of a 6-well plate in 2 ml
of complete IMDM 24 h before starting the titration;
2. Twenty-four hours later, thaw an aliquot of lentiviral particles
and prepare serial dilutions of the virus (e.g., from 10 −3 to
10 −7 ) in a final volume of 2 ml of culture medium containing
Polybrene at a final concentration of 8 μg/ml;
3. Aspirate the culture medium from the 6-well plate prepared
the day before and transduce the HEK293T cells by adding
1 ml of the different virus dilutions to each well;
4. The following day, approximately 18 h after the transduction,
add 1 ml of fresh complete IMDM;
5. Five days after transduction, collect transduced HEK293T
cells using Trypsin. Briefly, aspirate the culture medium, rinse
once with PBS 1×, and incubate with 500 ml/well of trypsin