A multiparametric, quantitative and high-throughput assay to quantify the influence of target size on phagocytic uptake

Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. It consists in the uptake of pathogenic or cellular targets larger than 0.5μm. For the biggest particles, the phagocytic process involves a massive reorganization of membrane and actin cytoskeleton as well as an important intracellular deformation, all in a matter of minutes. The study of the role of the size of objects in their phagocytosis has lead to contradictory results in the last decades. We designed a method using confocal microscopy, automated image analysis and databases for fast quantitative analysis of phagocytosis assays. It yields comprehensive data on the cells and targets geometric and fluorescence intensity parameters, automatically discriminates internalized from external targets, and stores the relationship between a cell and the targets it has engulfed. We used two types of targets, solid polystyrene beads and liquid lipid droplets, to investigate the influence of size on the phagocytic uptake of macrophages. The method made it possible, not only to perform phagocytic assays with functionalized droplets and beads of different sizes, but to use polydisperse particles to further our understanding of the role of size in phagocytosis. The use of monodisperse and polydisperse objects shows that while smaller monodisperse objects are internalized in greater numbers, objects of different sizes presented simultaneously are internalized without preferred size. Throughout results, the total surface engulfed by the cell appeared to be the main factor limiting the uptake of particles, regardless of their nature or size. A meta-analysis of the literature reveals that this dependence in surface is consistently conserved throughout cell types, nature of targets or activated receptors.