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.