Studying Phagocytosis by Live-Cell Scintillation Proximity Assay

  • Walter Stockinger
  • Axel Nohturfft
Part of the Methods in Molecular Biology book series (MIMB, volume 440)

Summary

Phagocytosis of microorganisms, senescent cells, apoptotic bodies, and effete tissue material is an important process in host defense and tissue homeostasis. A method is described to measure, in living macrophages, the kinetics of particle engulfment and lysosome/phagosome targeting. Plasma membranes or lysosomes are labeled with tritiated lipids, followed by exposure of cells to scintillant microbeads. Because of the short range of tritium β-particles, geometric factors, and the confinement of lipids to membranes, scintillation can only be elicited by tracer molecules in membranes immediately vicinal to the scintillant. When the plasma membrane.is labeled with [3H]cholesterol, a signal is produced on bead–cell contact and engulfment and then reaches steady state within 45 min. When lysosomes are labeled with nonhydrolyzable [3H]cholesterol oleyl ether, scintillation requires intracellular lysosome/phagosome attachment or fusion, and steady state is attained only after several hours. The live-cell scintillation proximity approach is useful for examining the effects of pharmacological and genetic manipulations on particle uptake and on lysosome/phagosome targeting.

Keywords

Cholesterol endocytosis endosomes lysosomes phagosomes plasma membrane 

Notes

Acknowledgments

This work was supported by funding from the Parseghian Medical Research Foundation and the National Institutes of Health (R01 DK59934).

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Walter Stockinger
    • 1
  • Axel Nohturfft
    • 2
  1. 1.Max F. Perutz LaboratoriesViennaAustria
  2. 2.Department of Molecular and Cellular BiologyHarvard UniversityCambridgeMA

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