Quantifying fluorescent nanoparticle uptake in mammalian cells using a plate reader

In keeping with the rapid expansion of nanoparticle applications, various tools are required to investigate how nanoparticles interact with biological entities. Many assays have been developed to measure the cellular uptake of nanoparticles, but so far most of the methods are laborious and often non-quantitative. Here we developed an easily accessible and robust quantitative measurement method of the level of cellular uptake of fluorescently labeled nanoparticles using a plate reader. In the experimental design, potential issues that could lead to measurement variation were identified and addressed. For example, the variation in fluorescence intensity of samples due to differences in cell number was normalized to optical density, which is a physical value corresponding to the cell number. Number of washings and sample handling temperature were optimized to minimize the interference by residual nanoparticles and possible efflux of nanoparticles from cells, respectively. The developed assay was demonstrated with the lymphocyte cell line Jurkat to measure the cellular uptake of fluorescently labeled 50 nm polystyrene beads, and its applicability was further confirmed with the lung carcinoma cell line A549 and another lymphocyte cell line RPMI8226.