Lentivirus stocks were produced as previously described with slight modifications (Cribbs et al., 2013 (link)). Human embryonic kidney 293FT cells (Invitrogen) were transfected using Lipofectamine 2000 (Invitrogen) with the expression of two helper plasmids: psPAX2 and pMD2.G. 10 µg transfer vector, 5 µg pMD2.G, and 5 µg psPAX2 of DNA were used per 10-cm plate. 48 h after transfection, the supernatants of four plates were pooled, centrifuged at 780 g for 5 min, filtered through a 0.45-µm pore size filter, and further centrifuged at 24,000 rpm for 2 h. The resulting pellet was resuspended in 100 µl of PBS. Lentivirus titration was performed with a p24 ELISA (Clontech).
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Lentivirus Production and Titration
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Cells
Elisa
Embryonic
Human
Kidney
Lentivirus
Lipofectamine 2000
Plasmids
Size
Titration
Transfection
Vector
Corresponding Organization : Nano and Advanced Materials Institute
Other organizations : Rutgers, The State University of New Jersey
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- Transfection of human embryonic kidney 293FT cells using Lipofectamine 2000 with the expression of two helper plasmids: psPAX2 and pMD2.G
- Lentivirus production and titration
- Amount of DNA used per 10-cm plate: 10 µg transfer vector, 5 µg pMD2.G, and 5 µg psPAX2
- Centrifugation at 780 g for 5 min
- Filtration through a 0.45-µm pore size filter
- Centrifugation at 24,000 rpm for 2 h
- Resuspension of the resulting pellet in 100 µl of PBS
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