Lysosensor yellow blue dnd

Tetramethylrhodamine ethyl ester (TMRE) (Invitrogen #T669) and MitoTracker green (Invitrogen #M7514) was both used at 100 nM in serum-free DMEM for 30 min at 37°C. MitoSox red (Invitrogen # M36008) was used at 5 μM in HBSS for 15 min at 37°C. LysoTracker deep red (Invitrogen #L12492) was used at 50 nM for 30 min. To detect cathepsin B activity, Magic red (Immunochemistry Tech. #937) was incubated with cells for 45 min (manufacturer’s protocol was followed). After staining, cells were washed and analyzed by flow cytometry. LysoSensor yellow/blue DND (Invitrogen #L7545) was used 2 μM for 5 min at 37°C. The ratiometric dye was excited by UV laser (355 nm) and fluorescence intensities through 585/42 (545 LP) and 450/50 filters were used to calculate relative pH by flow cytometry. LAMP1 was stained (Invitrogen #53-1071-82, 1:100) after permeabilizing the cells (BD Perm/Wash buffer) and analyzed by flow cytometry (BD LSR Fortessa instrument; BD FACSDiva and FlowJo software for analysis). LC3-GFP was measured after permeabilizing the cells with 0.05% saponin. Representative flow cytometry gating and histograms for LysoTracker deep red and Magic Red (cathepsin B activity) are shown in the Supplementary Information.