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High-sensitivity assessment of phagocytosis by persistent association-based normalization

Phagocytosis is measured as a functional outcome in many research fields but can be challenging to quantify accurately, with no robust method available for cross-laboratory reproducibility. Here, 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 (PAN), where first the multiplicity of prey (MOP) ratio needed to elicit half of the phagocytes to associate persistently (MOP50) is determined. MOP50 is then applied to normalize for experimental factors, with adhesion and internalization analyzed separately. We use THP-1 cells and different prey and opsonization conditions to compare the PAN method to standard ways of assessing phagocytosis, and find it better in all cases, with increased robustness, sensitivity, and reproducibility. The approach is easily incorporated into most existing phagocytosis assays and allows for reproducibly comparing results across experiments and laboratories with high sensitivity.