Lysosomal Dextran Labeling and Trafficking

Dextran loading and delivery to lysosomes were performed as previously described, with minor modifications^{49 (link),62 (link)}. Briefly, to pre-label lysosomes, HeLa^ORF3a-Strep cells, untreated or Dox-treated (1 µg/mL for 24 h), seeded on glass coverslips were incubated in phenol red-free complete DMEM media containing Alexa-Fluor 488-conjugated-dextran (green; Molecular Probes) for 12 h at 37 °C. Cells were washed once with phenol red-free complete DMEM and further incubated in phenol red-free complete DMEM containing Alexa-Fluor 568-conjugated-dextran (red; Molecular Probes) for the indicated time periods at 37 °C and 5% CO₂ in a cell culture incubator. At the end of the incubation period, the cells were washed with 1X PBS, fixed, and mounted as described earlier. The coverslips were immediately imaged using a confocal microscope. The colocalization of Alexa-Fluor 568-conjugated-dextran (red) with Alexa Fluor 488-conjugated-dextran (green) containing lysosomes was measured using the “JACoP” plugin of Fiji software.