Both recombinant lentiviruses and negative control were packaged in 293T cells in the presence of helper plasmids (pMDLg, pRSV-REV and pVSV-G) using TurboFect Infection Reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The infected cells were cultured at 37°C for 48 h and the virus was then collected for infection. Huh-7 and SK-Hep-1 were seeded in a 12-well cell culture plate, grown overnight at 37°C to achieve 30–40% confluence, and then infected with the virus in fresh medium containing 10 µg/ml polybrene. After 12 h of incubation, the culture medium was replaced with fresh medium and the infected cells were harvested at 72 h after infection and then used in subsequent experiments. All virus packaging and infection experiments were conducted in triplicate.
Turbofect infection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
TurboFect Infection Reagent is a transfection reagent designed for the efficient delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into a variety of cell types. It facilitates the uptake of these molecules into cells, enabling their expression or suppression for research purposes.
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Lab products found in correlation
2 protocols using turbofect infection reagent
1
Lentivirus Packaging and Infection Protocol
2
FoxO1 Knockdown in Bone Marrow-Derived Macrophages
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FoxO1-shRNA plasmids (Sigma, US) were kindly provided by Prof. Ping Gao from SCUT. The used FoxO1-shRNA sequence was 5′-CCGGCGGAGGATTGAACCAGTATAACTCGAGTTATACTGGTTCAATCCTCCGTTTTTG-3′ and 5′-CCGGCCGCCAAACACCAGTCTAAATCTCGAGATTTAGACTGGTGTTTGGCGGTTTTTG-3′. FoxO1-shRNA and NTC (negative control shRNA) plasmids paired with CMV△R8.91 (expressing 3 required HIV proteins) and PMD.G (expressing the VSV-G envelop proteins) plasmids were packaged for lentivirus with PEI (polyethyleneimine) in 293T cells. PEI was moved and replaced with normal culture medium 6 h later. Then, lentivirus was isolated from supernatants and cells after 48 and 72 h by 3000 × g 5 min centrifugation and filtration with 0.45 μm filter. Six-well plates cultured BMDMs were infected with 1 ml FoxO1-shRNA, and NTC lentivirus and 6 μl Turbofect infection reagent (Thermo, US) at the second day to sustain for 24 h, or infected cultured BMDMs with Ad5-CMV-Cre-GFP (Vigenebio, China) or control adeno-virus at 400 multiplicity of infection and sustain overnight. Then the macrophages were cultured in complete culture medium with 15% L929 culture supernatants for another 3 days.
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