Lipofectamine® 2000 Transfection Reagent

Protocol tips

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	Do not add antibiotics to the Transfection Medium

Protocol tips
Do not add antibiotics to the Transfection Medium

Publication protocol

Luciferase Assay
One TGF-β inducible Luciferase reporter construct contained the 378 bp of the α2(I) collagen (COL1A2) promoter and 58 bp of the transcribed sequence fused to the luciferase (Luc) open reading frame. The mutant COL1A2-LUC harbored a mutation in the Smad binding site of the TGF-beta response element within this promoter. These constructs did not contain the 5′ stem-loop. Both plasmids were kind gifts from Dr. Francesco Ramirez, Mount Sinai School of medicine and were described before . Another TGF-β responsive plasmid ((CAGA)MLP-Luc) contained 12 CAGA boxes cloned upstream of the initiator sequence of the adenovirus major late promoter (MLP) and was a kind gift of Dr. Peter ten Dijnke. This construct was also described before . Primary HLFs were seeded at a density of 10,000 cells/cm and cultured in 1% FBS. Cells were co-transfected using Lipofectamine 2000 (Invitrogen) with 0.2 µg of β-galactosidase plasmid and 0.8 µg of the Luciferase reporter plasmids described above. TGF-β1 (5 ng/ml) was added 24 h after transfections and luciferase readings were taken 16–24 h after that. To standardize the results for transfection efficiency, β-galactosidase activity was measured according to the standard protocol and luciferase activity was normalized to the β-gal activity.



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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Lipofectamine® 2000 Transfection Reagent below.

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