TY - JOUR
T1 - A novel assay for the quantification of internalized nanoparticles in macrophages
AU - Tian, Furong
AU - Prina-Mello, Adriele
AU - Estrada, Giovani
AU - Beyerle, Andrea
AU - Möller, Winfried
AU - Schulz, Holger
AU - Kreyling, Wolfgang
AU - Stoeger, Tobias
PY - 2008/11
Y1 - 2008/11
N2 - 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 μm, 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 this cell spreading area depends on FN concentration, and it is independent of particle concentration. An optimal condition of FN was found at 2.5×10-3 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.
AB - 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 μm, 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 this cell spreading area depends on FN concentration, and it is independent of particle concentration. An optimal condition of FN was found at 2.5×10-3 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.
KW - Internalized nanomaterials
KW - Materials science
KW - Mechanistic toxicology
KW - Quantitative assay
UR - http://www.scopus.com/inward/record.url?scp=57349160501&partnerID=8YFLogxK
U2 - 10.1080/17435390802504229
DO - 10.1080/17435390802504229
M3 - Article
AN - SCOPUS:57349160501
SN - 1743-5390
VL - 2
SP - 232
EP - 242
JO - Nanotoxicology
JF - Nanotoxicology
IS - 4
ER -