Measurement of Cholesterol Transfer from Lysosome to Peroxisome Using an In Vitro Reconstitution Assay

Low-density lipoproteins (LDLs) are taken up by the cell mainly through receptor-mediated endocytosis. LDL-derived cholesterol leaves lysosome and further transports to downstream organelles for specific cellular needs. We recently report that cholesterol transfers from lysosome to peroxisome through lysosome-peroxisome membrane contact (LPMC). Here, we use iodixanol density gradient centrifugation to isolate lysosomes and peroxisomes separately for the in vitro reconstitution of LPMC. We also apply ³H-cholesterol-labeled lysosomes and peroxisomes in vitro to measure ³H-cholesterol transfer through LPMC.