Phenol red free complete dmem media

To quantify Lysotracker uptake, cells were incubated in phenol red-free complete DMEM media (Gibco) containing 100 nM Lysotracker Red (LTR DND-99; Invitrogen) for 1 h at 37 °C. Post-incubation period, media was removed, and cells were trypsinized, washed, and resuspended in ice-cold 1X PBS and analyzed by flow cytometry. To measure the proteolytic activity of lysosomes, cells were incubated in phenol red-free complete DMEM media (Gibco) containing 20 µg/mL BODIPY FL-BSA (BioVision) for 2 h at 37 °C. Post-incubation period, media was removed, and cells were trypsinized, washed, and resuspended in ice-cold 1X PBS and analyzed by flow cytometry. Sample acquisition was done with BD FACS Aria Fusion Cytometer using BD FACS Diva software version 8.0.1 (BD Biosciences). Data analysis was done using BD FlowJo version 10.0.1.