Background

Monocytic lineage cells (monocytes, macrophages, and microglia) survey tissue for pathological threats, and act as hubs of the innate immune system. They exercise innate immune functions in numerous ways: by secreting cytokines and chemokines, acting through complement signaling pathways, and phagocytosing pathogens and debris. The extent of macrophage activation can tip the balance of immune response within the body during health and disease. In diseases such as cancer, the activation of monocytic cells can result in either pro- or anti- tumoral responses, which trigger different disease outcomes. Similarly, monocytic cells modulate the progression and remission of auto-immune and inflammatory diseases. It is therefore critical to characterize these cells and their responses, to identify steps which may be exploited therapeutically.

In vitro systems interrogate the functions of these innate immune cells in response to stimuli via specific assays. The phagocytosis assay is an essential tool in characterizing the magnitude of monocytic cell activation. Several variations, such as pulse-chase assay, particle loading, and/or bead loading, exist. Upon stimulation of the monocytic cell population with drugs, cytokines, or preconditioned cell media, the results of the assay report to what extent the cells have increased or decreased their ability to engulf particles from the environment, thus giving insight into how such stimuli may affect monocytic cells during health and disease in vivo.

Here we discuss some of the technical challenges associated with deriving high-quality data from standard phagocytosis assays. The variability of phagocytic activity of cells within a single sample makes this a particularly difficult function to assess. Most current studies count phagocytosed particles per cell, however, individual particles can be difficult to discern, and this measure can be misleading in cells that have higher baseline phagocytosis. Other studies simply analyze how many cells are phagocytosing (versus not), which gives a very broad and less quantitative picture of phagocytosis. Here, we have developed and propose the use of a new technical pipeline to collect and more sensitively analyze phagocytosis data. We then apply it to two different experiments for which macrophage phagocytosis is often assayed.