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Published on: August 18, 2018
A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester
Michael Miksa1, Hidefumi Komura, Rongqian Wu
1Center for Immunology and Inflammation, The Feinstein Institute for Medical Research, Manhasset, NY 11030, USA.
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.
Insights
This study introduces a new method using pHrodo dye to track the uptake of apoptotic cells by macrophages. This technique offers a simple and reliable way to study phagocytosis and its impact on immunity.
Area of Science:
- Immunology
- Cell Biology
Background:
- Incompetent clearance of apoptotic cells negatively impacts immunity.
- Accurate methods are needed to assess apoptotic cell internalization.
- pHrodo succinimidyl ester (SE) offers a pH-sensitive fluorescent label for detecting engulfed cells.
Purpose of the Study:
- To develop and validate a novel ex vivo assay for evaluating apoptotic cell internalization.
- To utilize pHrodo SE labeling for enhanced detection of phagocytosis.
- To assess the role of MFGE8 in apoptotic cell uptake by macrophages.
Main Methods:
- An ex vivo assay was designed using pHrodo-labeled apoptotic lymphocytes as prey.
- Tissue macrophages were labeled with anti-CD11b.
- MFGE8(-/-) macrophages were used to validate the assay's sensitivity to phagocytic defects.
Main Results:
- The pHrodo SE assay reliably detected the internalization of apoptotic lymphocytes.
- Stimulation with recombinant MFGE8 enhanced apoptotic lymphocyte uptake in splenic macrophages.
- Peritoneal macrophages demonstrated a compensatory uptake mechanism for delayed clearance.
Conclusions:
- The developed assay is a quick and reliable method for evaluating apoptotic cell internalization.
- This technique is valuable for phagocytosis studies using flow cytometry (FACS).
- The findings highlight MFGE8's role in phagocytosis and macrophage compensatory mechanisms.
