2.8. Lysosomal Membrane Integrity Assay

Neutral red (NR) assay was used to assess lysosomal membrane integrity. Cells were seeded into 96-well culture plates at a density of 10⁵ cells/well, differentiated into macrophages, and incubated with increasing concentrations of CDs for 24 h. At the end of the incubation period, culture medium was removed and cells were carefully washed with PBS. Complete culture medium containing NR (200 μL of a 100 μg/mL solution) was added to the cells for dye incorporation into intact lysosomes. After a 3-h incubation period, culture medium was removed and cells were lysed with 1% acetic acid solution containing 50% ethanol to release the incorporated dye. Absorbance of the resulting samples was read at 570 nm with a correction at 690 nm. Results were expressed as the percentage of the absorbance of treated cells relative to the absorbance of non-exposed control cells.