Real-Time and Quantitative Analysis of Macrophage Phagocytosis with RTCA eSight

Considering the critical roles that phagocytosis plays in microbial defense, wound healing, development,
and tissue homeostasis, it is not surprising that numerous assays have been developed to study phagocytosis in vitro. The primary legacy assays involve microscopy, flow cytometry, or spectrophotometry/spectrofluorimetry. In these assays phagocytes are typically incubated for a fixed period with particles (dextran, zymosan, E. coli) that are labeled to be constitutively fluorescent, followed by endpoint analysis. Quantification of phagocytosis by microscopy often requires cell fixation followed by manually counting the number of particles inside each cell, which is tedious and not amenable to
high throughput studies. Quantification of phagocytosis by spectrofluorimetry requires that uningested particles either be quenched or physically removed before analysis. Variations on these
assays, where particles are coupled to enzymes that are detectable using colorimetric substrates once cells have been fixed and permeabilized, still require a significant amount of hands-on time. The same is also true of flow cytometry‑based assays where phagocytes must be collected from the well bottom before they can be analyzed. In contrast to all these legacy methods, the eSight assay described here only requires mixing phagocytes with substrates; zero handing steps are subsequently required. Another distinguishing feature of this eSight phagocytosis assay is its continuous nature. Tracking phagocytosis in real time makes it possible to focus analyses on the most appropriate time regions.

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