Confocal Microscopy Slide Preparation
Slides were prepared for confocal microscopy with a standard fixing, washing, staining, and mounting protocol.9 PC-3 cells were grown for 48 hours on #1.5 glass coverslips after transfection and then excess media was removed before fixing for 10 minutes in 4% formaldehyde solution, followed by three washes with 1X PBS. Biotium Red CellBrite™ Cytoplasmic Membrane dye was diluted according to the manufacturer’s protocol (5 µL of cell staining solution in 1 mL of PBS) and then added to the coverslips, which were incubated for 10 minutes in the dark and then washed three more times. Two drops of Thermo Fisher NucBlue™ Live ReadyProbes™ Reagent (Hoescht 33342) were then added to the slips in 1 mL of PBS and incubated in the dark for 20 minutes. Finally, 10 µL of 1X PBS was used as mounting medium to mount the coverslips onto AmScope microscope slides (BS-72P) and sealed with clear nail polish. The same protocol was followed for primary T cells with 300 g for 4 minute centrifugation spins in between each step.
All images were obtained using a Leica TCS SP8 Four Channel Confocal Microscope with a 63X oil immersion lens. Lasers were set for 407 nm (DAPI), 488 nm (EGFP), and 633 nm (AlexaFluor 647). A three-sequence run was used to maximize reading while minimizing background and crosstalk. DAPI was set to PMT while EGFP and Alexa Fluor 647 were set to HyD. Images were taken using z-stacking from the bottom to the top of each cell.