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.