Development of innovative methods for phagocytosis quantification of microsized particles.

Inhaled particles exhibit variable toxicity levels which mainly depends on their physicochemical characteristics (size, morphology, surface chemical...). The relationship between the potential toxicity and the amount of phagocytosed particles is rarely investigated. The aim of this study was to develop a quantitative evaluation of phagocytosis using both direct and indirect methods in order to distinguish entirely engulfed microsized particles from those which are adherent to the cell surface. Fluorescent beads with variable and well-characterized sizes and surface coatings were incubated with RAW 264.7 macrophages at different concentrations and incubation times. The direct quantification of phagocytosis was based 1) on the measure of SSC parameter and fluorescence of intracellular beads 2) on the measure of pHrodo fluorescence, a specific sensor of acidic conditions. Indirect quantification of phagocytosed beads was based on cytoskeleton modifications evaluated by fluorescent phalloidin labeling. For all experiences, analysis were performed using both flow cytometry and confocal microscopy. The phagocytic cell percentage was greater for carboxylate 2 µm beads (60%) as compared to amine 1 µm beads (20%). Similarly SSC values were respectively 441 versus 131 for the two types of beads. Confocal imaging based on phalloidin labeling allowed visualization of entirely engulfed beads. Moreover the percentage of phagocytic cells increased with the beads/cell ratio and incubation duration. These results confirm that the phagocytosis level highly depends on physicochemical characteristics of particles