HUVECs at a density of 104 cells/well were seeded into 96-well plates and were then co-transfected with 100 ng NF-kB luciferase reporter plasmid (cat. no. YB003B; Shanghai Yu Bo Biological Technology Co., Ltd.) and 10 ng pGMLR-CMV luciferase Reporter plasmid (cat. no. 11558ES03; Shanghai Yeasen Biotechnology, Co., Ltd.) using Lipofectamine® 3000 (cat. no. L3000015; Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h post-transfection, cells were stimulated with 100 ng/ml LPS with or without tyrosol (0.5 mM or 1 mM) at 37˚C for 24 h. Luciferase activity was measured using the corresponding Dual-Luciferase Assay kit (Shanghai Yeasen Biotechnology, Co., Ltd.). Renilla luciferase was used as internal reference.
Lipofectamine 3000
Manufactured by Yeasen
Sourced in China
Lipofectamine 3000 is a cationic lipid-based transfection reagent used for the efficient delivery of DNA, RNA, or other nucleic acids into a variety of mammalian cell lines. It facilitates the uptake of these biomolecules into cells, enabling their use in various research applications.
Automatically generated - may contain errors
4 protocols using lipofectamine 3000
1
NF-kB Transcriptional Activity Modulation
2
Dual-Luciferase Assay for Interferon Signaling
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HEK293T cells plated in 24-well plates were cotransfected with reporter plasmid IFN-β-luc (125 ng), renilla luciferase plasmid pRL-TK (25 ng), together with vector alone or ADAP、RIG-I and MDA5. The transfections were performed by using Lipofectamine 3000 (Yeasen). 48 h later, cells were lysed and the luciferase activity was determined using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. The data were determined by normalization of the firefly luciferase activities to the renilla luciferase activities.
3
NF-κB Luciferase Reporter Assay
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Treated human T/C-28a2 cells were incubated for 24 hours followed by being transfected with NF-κB luciferase reporter (YEASEN, Shanghai, China) containing NF-κB response elements with the firefly luciferase gene inserted in the promoter region, with the promoterless-null Renilla construct (YEASEN, Shanghai, China) as the negative control. Lipofectamine 3000 (YEASEN, Shanghai, China) was used as the transfection reagent and the activity of luciferase and Renilla was measured using the Dual-Luciferase Reporter Assay System (Promega, Wisconsin, USA). Lastly, the luciferase activity of NF-κB was calculated after normalizing the value of luciferase activity with the activity of Renilla.
4
Regulation of E2F Transcription by ATO
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pGME2F-Luciferase reporter plasmid was purchased from Yeasen Biotechnology Company, the construct was transfected into KYSE-150 cells with Lipofectamine 3000®. 24 h after transfection, the KYSE-150 cells were treated with ATO (10 µM) for 48 hours. Then the cell lysates were collected, and luciferase activity was measured using a Yeasen Luciferase reporter gene assay kit (11401ES60).
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