UNIT 14.31 Measurement of Phagocytic Engulfment of Apoptotic Cells by Macrophages Using pHrodo Succinimidyl Ester
Published Online: 1 FEB 2013
Copyright © 2013 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Immunology
How to Cite
Aziz, M., Yang, W.-L. and Wang, P. 2013. Measurement of Phagocytic Engulfment of Apoptotic Cells by Macrophages Using pHrodo Succinimidyl Ester. Current Protocols in Immunology. 100:14.31:14.31.1–14.31.8.
- Published Online: 1 FEB 2013
- Published Print: FEB 2013
Considerable interest has emerged towards phagocytosis of apoptotic cells, due to its intricate molecular mechanisms and important regulatory functions in development, homoeostasis, and immune tolerance. Impaired clearance of apoptotic cells leads to immune-mediated disorders. Current quantification methods of the engulfment of apoptotic cells by macrophages are potentially flawed by several limitations. Adherent macrophage populations are overlaid with apoptotic targets in suspension and then co-cultured for a definite period, which may give rise to two different features: (1) engulfed and (2) non-engulfed macrophages that are surface-bound cell populations. Rigorous washing to dislodge surface-bound apoptotic cells before assessment of phagocytosis may lead to loss of phagocytes, thereby skewing the apparent magnitude of the overall phagocytic response. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. In this unit, we demonstrate the use of pHrodo-succinimidyl ester (SE), a pH-sensitive fluorescent dye, to label the apoptotic cells for monitoring the phagocytosis. After engulfment, the intensity of pHrodo light emission will be elevated due to the pH change inside of macrophages. The shift of pHrodo light emission can be detected by a flow cytometer or using a fluorescence microscope. Curr. Protoc. Immunol. 100:14.31.1-14.31.8. © 2013 by John Wiley & Sons, Inc.
- apoptotic cells;
- flow cytometry