To study endocytic trafficking, HK-2 and NRK-52E cells were, respectively, seeded into tissue culture-treated 35 mm plastic dishes from Ibidi (Glasgow, UK) or uncoated 35 mm glass-bottom dishes (No. 1.5 coverslip, 10 mm glass diameter) from MatTek (Ashland, USA). HK-2 and NRK-52E cells were seeded in 1.5 mL of CM at 60 000 and 75 000 cells per dish, respectively. After 24 h incubation at 37 °C with 5% CO2, cell culture medium was removed from each dish and replaced with CM containing wheatgerm agglutinin-Alexa Fluor 594 (WGA-AF594; 0.1 or 5 μg mL−1 for HK-2 and NRK-52E cells, respectively), as a physiological marker for endolysosomal content.16 (link) After 4 h, cell culture medium was removed and cells washed with PBS (3 × 1.5 mL). After the final wash, for the short chase experiment, cell culture medium was removed and replaced with freshly prepared filter-sterilised (0.22 μm) CM containing dextrin–OG, colistin–OG or dextrin–colistin-OG (5–10 μg mL−1 OG base) and leupeptin (200 μM). After a further 2 h incubation (pulse), cells were intensively washed with PBS, then, for the short chase experiment, cells were immediately incubated with Hoechst 33342 solution (1 μg mL−1) in CM (without phenol red or leupeptin) for 20 min before imaging. In contrast, for the long chase experiment, after incubation with WGA-AF594 for 4 h, cells were washed and 1.5 mL CM containing leupeptin (200 μM) was added to each well. After 1 h incubation, media was removed and replaced with CM containing filter-sterilised (0.22 μm) dextran-Alexa Fluor 488 (dextran-AF488, as control), dextrin–OG, colistin–OG or dextrin–colistin-OG and leupeptin (200 μM) for 2 h. Subsequently, cells were intensively washed with PBS then CM containing leupeptin (200 μM) was added and the plates incubated at 37 °C with 5% CO2 overnight. Finally, cells were washed with PBS then incubated with Hoechst 33342 solution (1 μg mL−1) in CM (without phenol red or leupeptin) for 20 min before imaging.
To study accumulation in mitochondria and Golgi in HK-2 cells, the same method was used, except that the WGA-AF594 step was omitted and at the end of the chase period, cells were incubated with Mitotracker™ Red FM (100 nM) or BODIPY™ TR ceramide (5 μM) in CM for 20 min before washing with PBS (3 × 1.5 mL). The protocols used here are summarised in Schemes S1 and 2.†
To study accumulation in mitochondria and Golgi in HK-2 cells, the same method was used, except that the WGA-AF594 step was omitted and at the end of the chase period, cells were incubated with Mitotracker™ Red FM (100 nM) or BODIPY™ TR ceramide (5 μM) in CM for 20 min before washing with PBS (3 × 1.5 mL). The protocols used here are summarised in Schemes S1 and 2.
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