Multiplexed Phagosomal Assays for the Detection and Quantification of Bidirectional Exchange Between the Phagolysosomal Lumen and Extracellular Space

The phagolysosome is an antimicrobial and degradative organelle that plays a key role in macrophage-mediated inflammation and homeostasis. Before being presented to the adaptive immune system, phagocytosed proteins must first be processed into immunostimulatory antigens. Until recently, little attention has been given to how other processed PAMPs and DAMPs can stimulate an immune response if they are sequestered in the phagolysosome. Eructophagy is a newly described process in macrophages that releases partially digested immunostimulatory PAMPs and DAMPs extracellularly from the mature phagolysosome to activate vicinal leukocytes. This chapter outlines approaches to observe and quantify eructophagy by simultaneously measuring several phagosomal parameters of individual phagosomes. These methods use specifically designed experimental particles capable of conjugating to multiple reporter/reference fluors in combination with real-time automated fluorescent microscopy. Through the use of high-content image analysis software, each phagosomal parameter can be evaluated quantitatively or semiquantitatively during post-analysis.