A novel assay for the quantification of internalized nanoparticles in macrophages

One of the most urgent requirements in nanotoxicology is a quantitative assessment of internalized nanomaterials in cells. We present an in vitro assay called 'max-flat' for the measurement of internalized particles in macrophages. Fluorescent polystyrene (PS) beads of diameters 1 mu m, 500 nm, 200 nm, 100 nm and 20 nm were employed. Different concentrations of fibronectin (FN) coated substrates were tested to achieve a maximal cell spreading area and minimal nucleus height, hence 'max-flat'. We found the cell spreading area depends on FN concentration, and it is independent of particle concentration. An optimal condition of FN was found at 2.5 x 10(-3) mg/ml, and it was selected for the max-flat assay to assess the internalization of PS beads. Cells under these conditions neither generate reactive oxygen species nor show noticeable differences in pro-survival/pro-apoptotic signals. Confocal images were employed for the max-flat assay and we set the interval scanning for a Z-stack as nucleus height divided by particle diameter. The max-flat assay provided a significantly higher number of internalized particles and the saturation is reached faster for nano-scale PS beads. We show how the proposed max-flat assay clearly outperform existing techniques by providing easier, more precise and far more reliable access to the number of internalized nanoparticles in macrophages.