PK-15 cells were plated at 1 × 106 per well in 6-well plates and cultured in DMEM complete medium for 24 h and then infected with PPV at a MOI of 1. After a further 24 h, the DMEM medium was removed. NS1 vector (4 μg/μL) and 10 μL Lipofectamine 3000 reagent (Beyotime Biotechnology, Shanghai, China) were separately mixed with 250 μL of the DMEM complete medium, allowed to stand at room temperature for 5 min, and then lightly mixed together and added to the cells for 20 min of culture at room temperature. Cells were then incubated with 500 μL mixed medium for 8 h at 37°C and recultured in complete medium for an additional 16 h at 37°C. Luciferase activities were detected using a TD-20/20 luminometer (Turner BioSystems, Inc., Sunnyvale, CA, USA). The pcDNA3.1A vector was transfected as mentioned above as a negative control, and different concentrations of FA were applied to the experimental groups.
Lipofectamine 3000 reagent
Manufactured by Beyotime
Sourced in China
Lipofectamine 3000 reagent is a transfection reagent used for the delivery of nucleic acids, such as plasmid DNA or RNA, into mammalian cells. It facilitates the uptake of these molecules into the cells, enabling their expression or silencing.
Automatically generated - may contain errors
Lab products found in correlation
Td 20 20 luminometer, by Promega (1 mentions)
Puromycin, by Beyotime (1 mentions)
Centrifuge, by Thermo Fisher Scientific (1 mentions)
Lv shspp1, by Genechem (1 mentions)
Lentiviral vectors small hairpin rna shrna, by Genechem (1 mentions)
Lv shcon, by Genechem (1 mentions)
Polybrene, by Merck Group (1 mentions)
293t cells, by Genechem (1 mentions)
2 protocols using lipofectamine 3000 reagent
1
Luciferase Assay for PPV Infection
2
Lentiviral shRNA Knockdown Protocol
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The lentiviral vectors (Lv)-small hairpin RNA (shRNA) based on a 3rd generation lentiviral system targeting PLAU (Lv-shSPP1), Lv-shYY1, and negative control (Lv-shCon) were purchased from Shanghai GeneChem Co., Ltd. Briefly, 1.5 µg shRNA vector and 1.5 µg package mix plasmids were transfected into 293T cells (cat. no. C6008; Beyotime Institute of Biotechnology) with Lipofectamine 3000® reagent (cat. no. L3000001; Thermo Fisher Scientific, Inc.) for 24 h at 37°C, and the medium was then harvested and the virus was extracted by 80,000 × g-ultracentrifugation at 4°C for 2 h in a centrifuge (Beckman Coulter, Inc.). For lentiviral transduction, the cells were seeded in six-well plates at a density of 50,000 cells/well. The lentiviral vector was added at a multiplicity of infection of 15, with 5 µl polybrene (Sigma-Aldrich; Merck KGaA) per well. After 72 h, cells infected with the lentiviral vectors were selected using 1 µg/ml puromycin (Beyotime Institute of Biotechnology). Knockdown efficiency was evaluated using RT-qPCR.
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