Alexa546 conjugated dextran

For endo-lysosome labelling, cells were pulsed with 50 to 100 μg/ml Alexa⁵⁴⁶-conjugated dextran (ThermoFisher) for 0.5 to 1 h, followed by 3× wash with PBS, and incubated with fresh medium for at least 1 h. To induce endo-lysosome remodelling, BMDMs and BMDCs were exposed to 100 ng/mL LPS from Salmonella enterica serotype minnesota Re 595 (Sigma-Aldrich, Oakville, ON), whereas RAW macrophages were incubated with 500 ng/mL for 2 h (unless otherwise stated). As noted earlier, we use the term ‘endo-lysosomes’ to reflect that this labelling method likely stains the spectrum between late endosomes, lysosomes, and their hybrids, endo-lysosomes. For pharmacological inhibition, cells were preincubated for 15 to 20 minutes with 100 nM torin1 (Tocris Bioscience, Minneapolis, MN), 10 μM cycloheximide (Bio-Shop), 1 μM LY2584702 (Selleck Chemicals, Houston, TX), or equivalent volume of vehicle. Cells were then imaged live (unless otherwise indicated) in complete medium. Lysosome were scored as tubules if their length was greater than 4 μm.

Lysosomes were labelled by incubating cells with 200 μg/mL Alexa⁵⁴⁶-conjugated dextran or with 200 μg/mL Alexa⁴⁸⁸-conjugated dextran (Thermo Fisher Scientific, Mississauga, ON) or with 2.5 mg/mL Lucifer yellow (Thermo Fisher Scientific, Mississauga, ON) for 2 h in complete media at 37°C in 5% CO₂. Cells washed with phosphate-buffered saline (PBS) and resupplied with complete cell-specific media for 1 h to chase the fluid-phase marker to lysosomes before pharmacological manipulation and live-cell imaging. We note that we use “lysosomes” to represent a potential mixture of late endosomes, lysosomes and endolysosomes [5 (link), 30 (link)]. Lysosomal calcium was labelled with Fluo-4AM 8 μM by pulsing for 45 min in complete media at 37°C in 5% CO₂, followed by washing with PBS and addition of complete media for 45 min to chase the marker to lysosomes.