Jurkat T Cell Growth and Transfection
Human T lymphocyte leukemia cells (Jurkat, clone E6-1, ATCC® TIB-152TM) were seeded in 24 well plates at a density of 2x105 cells/well in fetal bovine serum-containing RPMI-1640 media immediately prior to transfection. Polymer-mediated transfections were performed by mixing either branched PEI (MW = 25 kDa), linear PEI (MW = 10 kDa), jetPEI®, or TurboFectTM with plasmid DNA (1,000 ng DNA/well) and then incubating the mixture for 20 minutes at room temperature to form polyplexes. Transfections with Lipofectamine® LTX were performed according to the manufacturer’s protocol. Branched PEI (BP), linear PEI (LP), and jetPEI (JP) were all tested with 5:1 and 10:1 nitrogen:phosphate (N:P) ratios. N:P ratios could not be calculated for TurboFect and Lipofectamine LTX because their formulations are proprietary, so the following manufacturer-recommended volumes of each vehicle were tested for the delivery of 1 µg pDNA/well in a 24-well plate: “LipoHi” = 2.75 µL/well of Lipofectamine® LTX, “LipoLo” = 1.25 µL/well of Lipofectamine® LTX, and Turbofect = 6 µL/well of TurboFectTM. Polyplexes and lipoplexes were then added to the cells, which were subsequently incubated for 48 hours at 37°C. Cells were then either analyzed for EGFP expression via flow cytometry or lysed with 150 µL of cell culture lysis reagent/well (125 mM Tris-HCl pH 7.8, 10 mM DTT, 10 mM EDTA, 50% glycerol, and 5% Trition X-100) to quantify luciferase expression with a Promega Luciferase Assay Kit (#E1501).