High-Sensitivity Assessment of Phagocytosis by Persistent Association-Based Normalization

Abstract

Phagocytosis is measured as a functional outcome in many research fields, but accurate quantification can be challenging, with no robust method available for cross-laboratory reproducibility. In this study, we identified a simple, measurable parameter, persistent prey-phagocyte association, to use for normalization and dose-response analysis. We apply this in a straightforward analytical method, persistent association-based normalization, in which the multiplicity of prey (MOP) ratio needed to elicit half of the phagocytes to associate persistently (MOP 50 ) is determined first. MOP 50 is then applied to normalize for experimental factors, separately analyzing association and internalization. We use reference human phagocyte THP-1 cells with different prey and opsonization conditions to compare the persistent association-based normalization method to standard ways of assessing phagocytosis and find it to perform better, exhibiting increased robustness, sensitivity, and reproducibility. The approach is easily incorporated into most existing phagocytosis assays and allows for reproducible results with high sensitivity.

Opsonization by non-neutralizing antibodies can confer protection to SARS-CoV-2 despite Spike-dependent modulation of phagocytosis

“…Even though most of the experiments in this study are performed in vitro, we believe the effects we observe on phagocytosis could also be relevant in vivo, as the modulation effects occur already at relatively low antibody concentrations and would thus cover many physiological niches and scenarios. That an increased binding of antibodies to a prey results in reduced or blocked phagocytosis is in stark contrast to what is typically expected (28). It cannot be explained by specific monoclonal interactions, as it is seen across diverse monoclonals as well as in convalescent polyclonal samples.…”

A human monoclonal antibody bivalently binding two different epitopes in streptococcal M protein protects against infection

“…Phagocytosis is a receptor-mediated process where prey are internalized into phagosomes, followed by their maturation into acidic, hostile compartments (36). To investigate the ability of the antibodies to trigger phagocytosis, we used persistent association-based normalization (37) to study both the antibodies’ ability to increase phagocyte association as well as internalization. We incubated phagocytic THP-1 cells with pH-sensitive CypHer5-stained bacteria ( Supp Fig.…”

“…The phagocytosis experiments were performed using persistent association normalization (37). Prior to opsonization, the CypHer5E-and Oregon Green-stained SF370 bacteria were sonicated for up to 5 min (VialTweeter; Hielscher) to disperse any large aggregates of bacteria.…”

Lack of Opsonic Antibody Responses to Invasive Infections With Streptococcus dysgalactiae

“…The assay employed has previously been used for S. pyogenes and is technically difficult and highly dependent on the blood donor and has high interexperimental variation. Therefore, we wanted to further study if the typespecific antibodies increased the ability of phagocytes to bind and internalise S. dysgalactiae bacteria using high-sensitivity phagocytosis assessment (de Neergaard et al, 2019). In the phagocytosis assay we observed a small increase in the proportion of cells that associated with and internalised serum-incubated bacteria, but with no individual enhancement of phagocytic ability.…”

“…Serum from the patient, PW, which showed seroconversion between the second and the third episode, was assessed with respect to phagocytosis with an in-depth flow cytometric assay that takes into account both phagocyte association and internalisation (de Neergaard et al, 2019). Figure 4A visualizes the association capacity of the phagocytes with the percentage of phagocytes interacting (adhered or internalised) with at least one bacterium on the y-axis and the MOP on the x-axis.…”