Separation of magnetically isolated TNF receptosomes from mitochondria

We previously demonstrated that tumor necrosis factor receptor-1 (TNF-R1) initiates distinct TNF signaling pathways depending on the localization of the receptor. While TNF-R1 at the plasma membrane transmits proinflammatory and antiapoptotic signals, internalized TNF-R1 forms signaling endosomes (TNF receptosomes) that transmit proapoptotic signals. These findings were obtained by a novel technique for the isolation of morphologically intact endocytic vesicles containing magnetically labeled TNF-R1 complexes using a high-gradient, free-flow magnetic chamber. Since intact mitochondria appeared to be a major contaminating organelle in these preparations, we subsequently included a second purification step by iodixanol density centrifugation to obtain a mitochondria-free receptosome preparation.