Phagosome proteolysis was determined based on the dequenching of DQ-BSA/Alexa594 conjugated 3-μm silica beads (Kisker Biotech) as described by Sattler et al. (2013 (link)). Cells were seeded at 3 × 105 per well of a glass-bottomed 96-well plate (Greiner) in Lo-Flo medium (Formedium) in triplicate. After 1 h, 1.5 × 104 reporter beads were added to each well except controls, and plates were spun at 800 × g for 10 s to synchronize bead uptake. Cells were then washed twice with Lo-Flo to remove unengulfed beads before fluorescence measurements at 480/510 nm and 560/620 nm using a plate reader every 2 min. After subtraction of background fluorescence, DQ-BSA Signal was normalized for bead uptake using the Alexa594 signal.
Total proteolytic activity was performed in a similar way by adding the same reporter beads to cell lysates (Buckley et al., 2019 (link)). Samples of 4 × 107 cells/mL in 150 mM potassium acetate at pH 4.0 were lysed by two freeze–thaw cycles in liquid nitrogen. Cell debris was removed by centrifugation at 18,000 × g for 5 min at 4°C before 1.23 × 105 beads were added to 100 µl of supernatant in 96-well plates and measured as above. Cathepsin D levels were measured by Western blot using the previously published antibody (Journet et al., 1999 (link)).
Total proteolytic activity was performed in a similar way by adding the same reporter beads to cell lysates (Buckley et al., 2019 (link)). Samples of 4 × 107 cells/mL in 150 mM potassium acetate at pH 4.0 were lysed by two freeze–thaw cycles in liquid nitrogen. Cell debris was removed by centrifugation at 18,000 × g for 5 min at 4°C before 1.23 × 105 beads were added to 100 µl of supernatant in 96-well plates and measured as above. Cathepsin D levels were measured by Western blot using the previously published antibody (Journet et al., 1999 (link)).
