4.11. Lysosomal Membrane Permeabilization Assay

LMP was assessed as described previously [28]. Briefly, AM were seeded on 96-well plates at a density of 1 × 10⁵ cells per well and incubated at 37 ℃ for 4 h. Cells were then washed with PBS and incubated with 100 µL cytosol extraction buffer plus digitonin. The concentration of digitonin for optimal extraction of the cytosolic fraction was determined by titration. A 1:1 extracted cytosol and cathepsin reaction buffer was prepared, and the fluorescent intensity was read (ex/em: 400/489 nm) using a plate reader for 25 min with 45 s intervals. LDH activity was assessed following manufacturer’s instructions (Promega). Extracted cytosolic LDH activity was used as an internal control to which cytosolic cathepsin B was normalized. Cytosolic extract enzyme activities were calculated as a percent of total cell lysate activity in which 200 µg/mL digitonin was used to completely lyse the cells. The LMP assay was performed using AM incubated with 0–200 µg/mL of SiO₂ or NP at four different time points (0, 1, 2, and 4 h).