Lysosensor phrodo green am intracellular ph indicator

To assess lysosomal mass, microglia were stained with 100nM LysoTracker Red DND-99 (Invitrogen #L7528) for 30 mins. To assess lysosomal pH, microglia were washed with PBS and treated with 2μM LysoSensor Green DND-189 (Invitrogen #L7535) for 10 mins. Microglia were then washed with PBS three times, trypsinized, pelleted, resuspended in 100ul PBS, and stained with 0.1ug/ml DAPI for 5 min. Cells were again pelleted, washed three times in PBS, and resuspended in 100ul PBS at 60,000 cells/μL and analyzed using an Attune NxT flow cytometer. Cells were gated on DAPI to measure fluorescence intensity of LysoTracker and LysoSensor only in live cells. Calibration curve was prepared by incubating cells in LysoSensor pHrodo Green AM intracellular pH indicator (ThermoFisher Scientific #P35373) according to the manufacturer’s protocol.