Journal
JOURNAL OF IMMUNOLOGICAL METHODS
Volume 342, Issue 1-2, Pages 71-77Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2008.11.019
Keywords
pHrodo-SE; Phagocytosis; FACS; MFGE8
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Funding
- NIH [R01 GM057468, R01 GM053008, R01 AG028352]
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Apoptotic cell phagocytosis has recently raised considerable interest, particularly due to its intricate molecular mechanisms and negative immunologic impact of incompetent clearance of apoptotic cells. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. Labeling with pHrodo succinimidyl ester (SE), a pH-sensitive fluorescent dye, makes engulfed apoptotic cells detectable due to the increased post-phagocytic light emission. This is a valuable tool for phagocytosis studies via FACS. We designed an ex vivo assay, using apoptotic pHrodo-labeled lymphocytes as prey and anti-CD11b-labeled tissue macrophages. To demonstrate its validity of detecting internalized apoptotic lymphocytes, we used MFGE8(-/-) macrophages, known to have impaired phagocytic ability. Uptake of apoptotic lymphocytes was accelerated and enhanced in splenic macrophages after stimulation with recombinant MFGE8, while peritoneal macrophages were able to compensate for the delayed uptake. This novel assay is a quick and reliable method to evaluate the internalization of apoptotic cells. (C) 2009 Elsevier B.V. All rights reserved.
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