Pcl vsvg

293T cells were plated in triplicate in the absence of penicillin/streptomycin at a density of 250,000 per 6 well (Corning, 3516) 16-24 hours prior to transfection. Parental 293Ts were plated in 1 mL DMEM high glucose with 10% FBS whereas CSC293Ts were plated in 1 mL CSC media. Each well received 0.5 μg pGreenFire1-CMV (pGF1-CMV, transfer vector, System Biosciences), 0.25 μg ps-PAX2 (packaging vector, Addgene #12260), and 0.25 μg pCL-VSVG (envelope vector, Addgene #1733). Plasmids were diluted in 50 μL of DMEM without any supplements, vortexed, and then centrifuged for 5 seconds. 3 μL of FuGene HD (Promega, E2311) per well was added directly to the media in the DNA mixture. The tube was again vortexed and centrifuged for 5 seconds. The DNA and transfection reagent mixture was then incubated for 10 minutes at room temperature, and the entire solution in the tube subsequently added to a 6 well. Plates were then placed in a tissue culture incubator designated for lentivirus use.

293FT cells (ThermoFisher Scientific, Cat# R70007) were used to
generate lentiviral particles through co-transfection of the packaging
vectors pCMV-dR8.2 dvpr (Addgene, Plasmid # 8455) and pCl-VSVG (Addgene,
Plasmid # 1733) with the appropriate overexpression, shRNA, or other
plasmid. 293FT cells were seeded at a density of 1.2 million cells in DMEM,
high glucose (ThermoFisher Scientific, Cat# 11995073) in 10% Fetal Bovine
Serum (ThermoFisher Scientific, Cat# 26140079 with 1%
Penicillin-Streptomycin (ThermoFisher Scientific, Cat # 15140122). Cells
were incubated for 24 hours prior to transfection. Transfection was
performed using LipoD293^™ In Vitro DNA Transfection
Reagent (SignaGen Laboratories, Cat # SL100668) according to the
manufacturer’s instructions. Briefly, 5μg each of the
packaging plasmids and the plasmid of interest were combined into a tube,
the transfection reagent was diluted and added followed by a 15 minute
incubation. The transfection mixture was then added to the 293FT cells.
Media was changed after 16 hours. Virus was collected 48 hours after media
change and concentrated using the Lenti-X Concentrator (Clontech Takara Bio
USA, Cat # 631232) according to the manufacturers instructions. Viral
supernatants were centrifuged at 1,500 x g for 45 minutes and viral pellets
were frozen at −80°C for future use.