Lysosome Enrichment Using Superparamagnetic Iron Oxide Nanoparticles (SPIONs)

Abstract

Lysosomes are dynamic and low-abundant organelles, and their enrichment is essential to facilitate a comprehensive biochemical characterization. Several methods are available to achieve this goal, such as density gradient centrifugation, immunoprecipitation, and superparamagnetic iron oxide nanoparticles (SPIONs). Compared to the other approaches, SPIONs present several advantages, as they enable the fast enrichment of intact lysosomes at high yields and purity without the need for the expression of tagged proteins. This protocol describes an optimized workflow for the enrichment of lysosomes from mammalian cells using SPIONs. We describe cell culture and treatment conditions tailored toward commonly utilized cell lines, approaches for cell rupture and lysosome enrichment, and downstream analyses by enzymatic assays and western blotting to assess the success of lysosome enrichment.

Keywords: Dextran; Endocytosis; Lysosomes; SPIONs; Superparamagnetic nanoparticles; β-hexosaminidase assay.