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

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 (MOP50) is determined first. MOP50 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.