Objectives
• Discover how the various cells of the adaptive immune system respond to opsonization
• Learn to evaluate phagocytosis in single cells using flow cytometry
• Determine optimal E. coli-to-cell ratios and assay conditions for optimal data interpretability
• Follow along with step-by-step gating instructions for acquiring multiparameter data across the cell types analyzed
Human health relies on several inborn protections from illness-inducing organisms. These defense mechanisms take several forms – including physical barriers, such as the skin, mucous membranes, and their cilia, the inflammatory cascade, as well as a cadre of cellular host-defense systems – collectively known as the immune system. Cellular immunity is divided into two classes, the innate immune system and the adaptive immune system. The innate immune system is tuned to providing a quick, knee-jerk, nonspecific response to invading organisms, and it relies heavily on neutrophil granulocyte-, and monocyte-mediated recognition and clearance of the threat. Diminished phagocytic activity in these cells can render the immune system less efficient at squelching infection. Understanding how key immune cell populations respond to and protect against non-self cells is essential to better understanding how we may exploit those cells for human health.
In the innate immune system, phagocytosis is the primary process by which cells identify, isolate, and destroy invaders by engulfing and digesting them. Among the phagocytic cells of the immune system, neutrophilic granulocytes (neutrophils) are the most abundant type of leukocyte (white blood cells) in mammalian circulation and the first line of defense against invading microorganisms.
Of the leukocytes, monocytes are the largest and are capable of differentiating into macrophages Opsonization adorns invading cells, like bacteria, with molecular pro-phagocytosis signals, enhancing their uptake and clearance. This process specifically tags foreign cells with opsonin molecules, such as circulating antibodies or complement proteins. Analogous to molecular “handles,” opsonins strengthen binding between immune cells
and their target cells to increase their visibility to the immune system, increasing the likelihood of phagocytosis (Figure 1). Opsonization is, indeed, essential to the efficient functioning of the innate immune system, and this application note demonstrates the important role that opsonisation plays in phagocytosis.
There are many effective methods to evaluate phagocytosis, such as enzyme-linked immunosorbent assay (ELISAs) and Western blotting, however, these assays look at cell populations as a whole and require substantial hands-on time. Flow cytometry enables a detailed analysis of the progression of phagocytosis in a population of cells, bringing the advantage of high-throughput, single-cell analysis. Multicolor flow cytometry offers an even higher throughput, while increasing the number of targets analyzed in a single run.1 Fluorescent labeling of cell-surface markers enables the quantification of specific immune cell types.
