Assessment and quantification of crystal-induced lysosomal damage

Lysosomes are organelles that degrade endocytosed, phagocytosed, or autophagocytosed materials. Lysosomal degradation of engulfed material is a highly controlled mechanism, which is vital in the control of infection, recycling of cellular organelles, and the breakdown of larger material. Following ingestion, lysosomes acidify and mature, leading to the activation of a range of proteolytic enzymes. Lysosomes are considered stable organelles that separate their highly hydrolytic enzymes from the cytosol to prevent digestion of self-molecules and host cell damage. Some substances, mostly of a particulate or aggregated nature, are able to damage lysosomes. Lysosomal damage and rupture, with subsequent release of lysosomal content into the cytosol can have dramatic consequences for the cell, such as the induction of cell death or activation of the NLRP3 inflammasome. In this chapter, we provide methods for the induction, assessment, and quantification of lysosomal damage by flow cytometry and confocal microscopy.