A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester

Michael Miksa, Hidefumi Komura, Rongqian Wu, Kavin G. Shah, Ping Wang

Research output: Contribution to journalArticle

184 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)71-77
Number of pages7
JournalJournal of Immunological Methods
Volume342
Issue number1-2
DOIs
StatePublished - Mar 15 2009

Keywords

  • FACS
  • MFGE8
  • Phagocytosis
  • pHrodo-SE

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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