Fluorescent Plasmid Transfections
Transfections of Mirus Bio™ Label IT™ fluorescein-labeled plasmid (MIR 7906) were conducted by mixing 2.75 µL Lipofectamine with 1 µg pDNA for each well of 2x105 Jurkats and CD3+primary T cells, while wells with 1x105 PC-3 cells were transfected with 1 µL Lipo and 500 ng pDNA. Jurkat and primary T cells were both cultured and transfected in serum-free X-VIVO™ 15 media, while PC-3 cells were cultured and transfected in RPMI 1640 media supplemented with 10% FBS. Following transfection, all cells were incubated for 24 hours and then spun down at 300 g for 4 minutes to remove excess pDNA in the media. The cells were then resuspended in trypsin-EDTA solution and incubated for 10, 20, or 30 minutes to disrupt interactions between cell surface proteins and lipoplexes. Once the incubation was complete, the trypsin was quenched with serum, the cells were spun down again, resuspended in phosphate buffered saline (PBS) that was supplemented with 10% FBS, and then analyzed for fluorescein labeling via flow cytometry. The same protocol was followed for the PC-3 cells, but with the addition of an initial trypsinization to detach the cells from the culture plate.