Optimizing transfection of primary human umbilical vein endothelial cells using commercially available chemical transfection reagents.
Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays.
Hunt
Michelle A
MA
Angiogenesis and Cancer Research Group, University of Otago, Christchurch, New Zealand.
Currie
Margaret J
MJ
Robinson
Bridget A
BA
Dachs
Gabi U
GU
D002470:Cell Survival; D042783:Endothelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D011933:Reagent Kits, Diagnostic; D014162:Transfection / Q000379:methods*; D014471:Umbilical Veins / Q000166:cytology*
D007202:Indicators and Reagents; D011933:Reagent Kits, Diagnostic; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins