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. 2022 Nov 23;12(1):20146.
doi: 10.1038/s41598-022-24480-3.

Quantifying fluorescent nanoparticle uptake in mammalian cells using a plate reader

Hye Ji Shin  1 , Minjeong Kwak  2 , Sihwa Joo  3 , Ji Youn Lee  4
Affiliations

Quantifying fluorescent nanoparticle uptake in mammalian cells using a plate reader

Hye Ji Shin et al. Sci Rep. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Overall experimental procedure for nanoparticle uptake analysis including cell seeding, nanoparticle treatment, cell harvesting and washing, and plate reading and analysis.
Figure 2
Figure 2
(A) Correlation between cell number and optical density measured at 600 nm and (B) FITC intensity change by repeated washing of Jurkat cells.
Figure 3
Figure 3
Quantification of nanoparticle uptake by Jurkat cells for biological replicates. The scatter plots show three biological replicates of Jurkat cells treated with different concentrations of PSNP50. (A) FITC intensity measured by a plate reader, (B) optical density measured at 600 nm by a plate reader, (C) normalized FITC intensity by the optical density, and (D) FITC intensity measured by a flow cytometer.
Figure 4
Figure 4
Quantification of nanoparticle uptake by Jurkat cells in multiple experiments. The scatter plots show three different sets of experiments with Jurkat cells treated with different concentrations of PSNP50. (A) Before and (B) after the normalization of FITC intensity with absorbance at 600 nm.
Figure 5
Figure 5
Scatter plots showing the correlation between cell number and optical density measured at 600 nm for (A) A549 and (B) RPMI8226 cells. FITC-PSNP50 uptake results of different sets of experiments with (C) A549 and (D) RPMI8226 cells.
See this image and copyright information in PMC

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