Determination of intracellular localization of fluorescent nanoparticles v1

This protocol investigates cellular uptake mechanisms and intracellular localization of polystyrene nanoparticles (PS-NPs) in HEK 293 cells. Confocal microscopy is used to monitor time-dependent internalization, clathrin-mediated endocytosis inhibition, and vesicular trafficking. It quantitatively analyzes nanoparticle internalization and spatial distribution relative to the nucleus using image segmentation and computational analysis, and evaluates how serum protein adsorption (protein corona formation) modifies PS-NP surface charge, size, and cellular uptake. Results: Both positive and negative PS-NPs entered cells primarily by clathrin-mediated endocytosis and were rapidly trapped by lysosomes. However, amine modified positive NPs were more uptaken than negative ones.