Simultaneous Analysis of Multiple Lumenal Parameters of Individual Phagosomes Using High-Content Imaging

The biochemical processes within the phagosomes of macrophages and dendritic cells are essential to immunity and homeostasis. Measurable properties of the phagosomal lumen include: assessment of various hydrolytic activities, reduction and oxidation events, pH, ion concentrations, and electrochemical gradients. These often-interdependent phagosomal features are commonly evaluated individually, hindering the analysis of the biochemical relationship between these factors within the same phagosome. In addition, the ability of phagosomes within the same cell to behave autonomously is becoming more evident, thus highlighting the need for a technique capable of multiplex analyses of phagosomal lumenal chemistries at the single phagosome level. In this chapter, we outline an approach that is capable of simultaneously measuring multiple phagosomal parameters of individual phagosomes by utilizing specifically designed experimental particles with multiple reporter fluors, in combination with real-time fluorometric measurement via automated microscopy. Subsequent analysis using high-content image analysis software enables each phagosomal parameter to be evaluated in a quantitative or semi-quantitative manner. This approach facilitates investigation of the complex relationship between phagosomal properties in a population of macrophages in real time, at the level of individual phagosomes.