Similar to the immunofluorescence methods above, OVISE Cas9 stable cells were plated at 10,000 cells per well in μ-Slide 8 Well coated chamber slides (IBIDI, cat# 80826) and simultaneously infected with lentivirus expressing sgRNAs. 24 hours after infection, cells were selected with 2ug/mL puromycin for 2 days and then replaced with RPMI-1640 without phenol red supplemented with 10% FBS. Live cells were incubated with dyes and imaged 5 days after infection.
For dye staining, LysoTracker Red DND-99 (Invitrogen cat#: L7528) was resuspended as a 1 mM stock in DMSO. Cells were stained in phenol-red free RPMI growth media 50 nM Lysotracker for 45 min at 37C in the dark. The dye was then washed out once with dye free growth media and then incubated with 1 ug/mL Hoechst 33342, (Invitrogen cat#: H3570) in phenol red free growth media at 37C for 30 min and remained in Hoechst containing media during imaging. Cells were imaged immediately after on a Nikon Eclipse Ti microscope with a Yokogawa Life Sciences CSU-W1 spinning disc confocal system.
For dye staining, LysoTracker Red DND-99 (Invitrogen cat#: L7528) was resuspended as a 1 mM stock in DMSO. Cells were stained in phenol-red free RPMI growth media 50 nM Lysotracker for 45 min at 37C in the dark. The dye was then washed out once with dye free growth media and then incubated with 1 ug/mL Hoechst 33342, (Invitrogen cat#: H3570) in phenol red free growth media at 37C for 30 min and remained in Hoechst containing media during imaging. Cells were imaged immediately after on a Nikon Eclipse Ti microscope with a Yokogawa Life Sciences CSU-W1 spinning disc confocal system.