Alexafluor 568 conjugated dextran

For tracing the endocytic pathway, fluid phase markers were used. HeLa cells were transfected with LAMP1-GFP, infected as described and 4 h p.i. cells were incubated with 100 µg x ml⁻¹ AlexaFluor 568-conjugated dextran, (molecular weight 10,000, Molecular Probes) for 3 h, washed, and incubated for the rest of the experiment with dextran-free medium. Later cells were processed for imaging.

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.

For LysoTracker Red staining, late L3 stage larval garland nephrocytes were dissected in cold Shields and Sang M3 medium (Sigma Aldrich), and then incubated in medium containing LysoTracker Red (1:1000, Thermo Fisher) for 5 min at room temperature (RT). Samples were rinsed 3 times and photographed immediately. For dextran uptake assay, late L3 stage larval garland cells were dissected in ice cold M3 medium, and then incubated in medium supplemented with Alexa Fluor 568 conjugated dextran (1 mg/ml, fixable, 10000 Da, Molecular Probes) for 5 min at RT, rinsed 3 times and fixed with 4% formaldehyde (FA) in PBS (30 min at RT). Nuclei were counterstained with DAPI in both cases. Pictures were taken on a microscope (AxioImager.Z1; Carl Zeiss) equipped with a grid confocal unit (ApoTome1; Carl Zeiss), using 40×, 0.75 NA (air), and 63×, 1.4 NA (oil) objectives in Lysotracker and dextran experiments, respectively, a CCD camera (AxioCam MRm; Carl Zeiss), and AxioVision software (Carl Zeiss).