jetPEI® DNA transfection, HTS application

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always add your plasmid to the buffer before adding jetPEI

Upstream tips
Always add your plasmid to the buffer before adding jetPEI

Publication protocol

2.6.4. Luciferase expression assay
Prior to transfection, either primary neonatal human dermal fibroblast cells (HDFn, Invitrogen, Carlsbad, CA) or rat mesenchymal stem cells (RMSC, Invitrogen, Carlsbad, CA) were plated on 24-well plates at a density of 100,000 cells per well, with approximately 70% confluency. HDFn cells were incubated in Medium 106, supplemented with 2% FBS, hydrocortisone (1 μg/mL), human epidermal growth factor (1 ng/mL), basic fibroblast growth factor (3 ng/mL), and heparin (10 μg/mL), for 24 h at 37 °C in a 5% CO2 environment. RMSC were incubated in Mesanpro RS medium (Invitrogen) supplemented with 2% FBS. For both cell lines, medium was changed 30 minutes prior to transfection. Stock solutions at N/P 20 for each polymer were prepared and diluted to lower N/P ratios (10, 7) so that equal volume of polymer solution (V = 13.33 μL) and pDNA ([pDNA] = 0.075 mg/mL, Vt = 13.33 μL) could be combined to form the polyplex solution for each well (total pDNA per well = 1 μg). Positive controls - Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA), jetPEI™ (Polyplus Transfection™, New York, NY), and Glycofect™ (Techulon Inc., Blacksburg, VA) – were formulated with pDNA based upon their recommended protocols. Solutions of transfection reagent-pDNA (gWiz-Luciferase, Aldevron, Fargo, ND) complexes for each N/P were added in triplicate to the corresponding wells. Plates were swirled to ensure homogenous solution formation and incubated for 4 h, after which more medium (500 μL) was added to each well. Cells were incubated for additional 20 h, followed by a medium change and 24 h of additional incubation time (48 h total). Medium was removed from wells and cells were lysed in 100 μL Cell Lysis Buffer (Promega, Madison, WI). Cell lysates were deposited on 96-well plates and analyzed for luciferase activity. Data was reported in Relative Light Units (RLU) and normalized to cells only control.

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Manufacturer protocol

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

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