Separation of magnetically isolated TNF receptosomes from mitochondria

Methods Enzymol. 2014:535:327-49. doi: 10.1016/B978-0-12-397925-4.00019-5.

Abstract

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.

Keywords: Endosomes; Magnetic separation; Mitochondria; Receptosomes; TNF-receptor 1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Fractionation / methods*
  • Cell Line, Tumor
  • Centrifugation, Density Gradient
  • Endosomes / metabolism
  • Humans
  • Immunomagnetic Separation
  • Mitochondria / metabolism*
  • Transport Vesicles / metabolism*
  • Tumor Necrosis Factor-alpha / isolation & purification
  • Tumor Necrosis Factor-alpha / metabolism*

Substances

  • TNF protein, human
  • Tumor Necrosis Factor-alpha