jetPEI® DNA transfection, HTS application

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

SH-SY5Y cells were seeded in 96-well plates at a density of 9,000 cells per well and grown for 12 h. Afterwards, a total of 250 ng of DNA was transfected into each well, adjusting to the following conditions: 250 ng of pcDNA3 plasmid (blanks); 25 ng of Renilla, 25 ng of CMVLuciferase Vector and 200 ng of pcDNA3 (controls); and 25 ng of Renilla, 25 ng of BACE1-5’UTR CMV-Luciferase construct, 25 ng of BACE1-mutated-5’UTR CMV-Luciferase construct (the mutation consists in the change of the 4 ATG triplets of the 5’UTR by 4 TTG; Genscript) and 200 ng of pcDNA3 (test samples). Cells were transfected using JetPEI transfection reagent (PolyPlus, Illkirch, France) for 2 h. The medium was changed and the cells incubated for 10 h to allow gene expression. Cells were treated with 100 µM of the inhibitor of p-eIF2 phospatase PP1 salubrinal (Sal003; Tocris) or the NO donor sodium nitroprusside (SNP; Sigma) for 1 h. 100 100 µM SNP provides a flow of 100 µM/min of NO (10). Cells were lysed and luciferase and Renilla activities were measured by using the Dual-Glo™ Luciferase Assay System (Promega, Wisconsin, USA) following manufacturer’s instructions and luminescence was read using a luminometer (Fluostar OPTIMA, BMG Labtech).

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Manufacturer protocol

Download the product protocol from Polyplus transfections for jetPEI® DNA transfection, HTS application below.

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