Lysosome Membrane Permeabilization Assay

Lysosome membrane permeabilization (LMP) was assessed using methods modified from Aits et al. (2015) (link) and as described previously by our laboratory (Jessop et al., 2017 (link)). BMDM were plated in 24 well plates at a density of 2×10⁵ cells per well. Cells were treated with or without HCQ (25 μM) and with or without cSiO₂ (50 μg/mL). Cells were washed twice with PBS and placed on ice. BMDM were then incubated with 200 μL of cytosol extraction buffer, which consisted of 250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.5 mM pefabloc (Sigma-Aldrich cat. 76307-100mg), pH 7.5, and digitonin (15 μg/mL ) (Sigma-Aldrich cat. D141-100MG), for 15 min on ice with rocking. The concentration of digitonin for optimal extraction of the cytosolic fraction was determined by titration. β-N-acetylglucosaminidase (NAG) activity was measured by adding 30 μL cytosolic extract to 100 μL of NAG reaction buffer (0.2 M sodium citrate, pH 4.5 with 300 μg/mL 4-methylumbelliferyl-2-acetamido-2-deoxy-β-D-glucopyranoside (Sigma-Aldrich cat. 37067-30-4) and assessed on a plate reader (20 min; 45s intervals; 356 nm excitation; 444 nm emission). Extracted cytosolic LDH activity was measured as described above and used as a control to which the NAG activities were normalized.