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Tian, F. ; Prina-Mello, A.* ; Estrada, G. ; Beyerle, A. ; Möller, W. ; Schulz, S. ; Kreyling, W.G. ; Stöger, T.

A novel assay for the quantification of internalized nanoparticles in macrophages.

Nanotox. 2, 232-242 (2008)

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Open Access Green as soon as Postprint is submitted to ZB.

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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 1µm, 500 nm, 200 nm, 100 nm and 20 nm were employded. 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 conditon 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.

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