Detection of Silver and TiO2 Nanoparticles in Cells by Flow Cytometry

  • Robert Martin ZuckerEmail author
  • William K. Boyes
Part of the Methods in Molecular Biology book series (MIMB, volume 2118)


Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. A FACSCalibur™ flow cytometer and a Stratedigm S-1000 flow cytometer were used to measure changes in light scatter from cells after incubation with either silver nanoparticles (AgNP) or TiO2 nanoparticles. Within the range of between 0.1 μg/mL and 30 μg/mL the nanoparticles caused a proportional increase of the side scatter and decrease of the forward scatter intensity signals. At the lowest concentrations of TiO2 (ranging between 0.1 μg/mL and 0.3 μg/mL), the flow cytometer can detect as few as 5–10 nanoparticles per cell. The influence of nanoparticles on the cell cycle was detected by nonionic detergent lysis of nanoparticle incubated cells that were stained with DAPI or propidium iodide (PI). Viability of nanoparticle treated cells was determined by PI exclusion. Surface plasmonic resonance (SPR) was detected primarily in the far-red fluorescence detection channels after excitation with a 488 nm laser.

Our results suggest that the uptake of nanoparticles within cells can be monitored using flow cytometry. This uptake of nanoparticle data was confirmed by viewing the nanoparticles in the cells using dark-field microscopy. The flow cytometry detection of nanoparticles approach may help fill a critical need to assess the relationship between nanoparticle dose and cellular toxicity. Such experiments using nanoparticles could potentially be performed quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.

Key words

Nanoparticles Flow cytometer Cytometry Toxicology Nanotoxicity Side scatter Plasmonic surface resonance 



The manuscript was edited by Enrico Ferrari and Mikhail Soloviev. Thanks are extended to Laura Degn, for her helpful comments and to Wiley & Sons and Springer books for allowing us to reproduce their figures in the publication.

Government Disclaimer: The research described in this chapter has been supported by the US Environmental Protection agency. It has been subjected to agency review and does not necessarily reflect the views of the agency, and no official endorsement should be inferred. The mention of trade names or commercial products does not constitute endorsement or recommendation for use.


  1. 1.
    Maynard AD, Aitken RJ, Buts T et al (2006) Safe handling of nanotechnology. Nature 444:267–269CrossRefGoogle Scholar
  2. 2.
    Kauffman M, Rose J, Bottero JY et al (2009) Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. Nat Nanotechnol 4:634–641CrossRefGoogle Scholar
  3. 3.
    Boyes WK, Thornton BLM, Al-Abed SR et al (2017) A comprehensive framework for evaluating the environmental health and safety implications of engineered nanomaterials. Crit Rev Toxicol 47:767–810CrossRefGoogle Scholar
  4. 4.
    Nohynek GJ, Lademann J, Ribaud C et al (2007) Grey goo on the skin? Nanotechnology, cosmetic and sunscreen safety. Crit Rev Toxicol 37:251–277CrossRefGoogle Scholar
  5. 5.
    Ortenzio J, Degn L, Goldstein-Plesser A et al (2019) Determination of silver nanoparticle dose in vitro. NanoImpact 14:100156CrossRefGoogle Scholar
  6. 6.
    Salzman GC (2001) Light scatter: detection and usage. Curr Protoc Cytom 9:1.13.1–1.13.8Google Scholar
  7. 7.
    Shapiro HM (2003) Practical flow cytometry, 4th edn. John Wiley & Sons, Inc., Hoboken, NJCrossRefGoogle Scholar
  8. 8.
    Shapiro HM (2001) Optical measurements in cytometry: light scattering, extinction, absorption, and fluorescence. Methods Cell Biol 63:107–129CrossRefGoogle Scholar
  9. 9.
    Steen HB (2004) Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles. Cytometry A 57:94–99CrossRefGoogle Scholar
  10. 10.
    Zucker RM, Elstein KH, Easterling RE et al (1988) Flow cytometric discrimination of nuclei by right angle scatter. Cytometry 9:226–231CrossRefGoogle Scholar
  11. 11.
    Nüsse M, Jülch M, Geido E et al (1989) Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements. Cytometry 10:312–319CrossRefGoogle Scholar
  12. 12.
    Zucker RM, Perreault SD, Elstein KH (1992) Utility of light scatter in the morphological analysis of sperm. Cytometry 13:39–47CrossRefGoogle Scholar
  13. 13.
    Giaretti W, Nüsse M (1994) Light scatter of isolated cell nuclei as a parameter discriminating the cell-cycle subcompartments. Methods Cell Biol 41:389–400CrossRefGoogle Scholar
  14. 14.
    Zucker RM, Massaro EJ, Sanders KM et al (2010) Detection of TiO2 nanoparticles in cells by flow cytometry. Cytometry A 77:677–685CrossRefGoogle Scholar
  15. 15.
    Zucker RM, Daniel KM, Massaro EJ et al (2013) Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence. Cytometry A 83:962–972PubMedGoogle Scholar
  16. 16.
    Prasad RY, Simmons SO, Killius MG et al (2014) Cellular interactions and biological responses to titanium dioxide nanoparticles in HepG2 and BEAS-2B cells: role of cell culture media. Environ Mol Mutagen 55:336–342CrossRefGoogle Scholar
  17. 17.
    Prasad RY, Wallace K, Daniel KM et al (2013) Effect of treatment media on the agglomeration of titanium dioxide nanoparticles: impact on genotoxicity, cellular interaction, and cell cycle. ACS Nano 7:1929–1942CrossRefGoogle Scholar
  18. 18.
    Suzuki H, Toyooka T, Ibuki Y (2007) Simple and easy method to evaluate uptake potential of nanoparticles in mammalian cells using a flow cytometric light scatter analysis. Environ Sci Technol 41:3018–3024CrossRefGoogle Scholar
  19. 19.
    Zucker RM, Daniel KM (2012) Microscopy imaging methods for the detection of silver and titanium nanoparticles within cells. In: Soloviev M (ed) Nanoparticles in biology and medicine. Methods Mol biol, vol 906. Humana Press, Totowa, New Jersey, pp 483–496CrossRefGoogle Scholar
  20. 20.
    Zucker RM, Ortenzio JN, Boyes WK (2016) Characterization, detection, and counting of metal nanoparticles using flow Cytometry. Cytometry A 89:169–183CrossRefGoogle Scholar
  21. 21.
    Zucker RM, Ortenzio J, Degn LL et al (2019) Biophysical comparison of four silver nanoparticles coatings using microscopy, hyperspectral imaging and flow cytometry. PLoS One 14:e0219078CrossRefGoogle Scholar
  22. 22.
    Zucker RM (2008) Flow cytometry quality assurance. In: Resch-Genger U (ed) Standardization and quality assurance in fluorescence measurements II. Springer series on fluorescence, vol 6. Springer, Berlin, HeidelbergGoogle Scholar
  23. 23.
    Zucker RM, Fisher NC (2013) Evaluation and purchase of an analytical flow cytometer: some of the numerous factors to consider. Curr Protoc Cytom 63:1.28.1–1.28.11Google Scholar
  24. 24.
    van der Pol E, van Gemert MJ, Sturk A et al (2012) Single vs. swarm detection of microparticles and exosomes by flow cytometry. J Thromb Haemost 10:919–930CrossRefGoogle Scholar
  25. 25.
    Zucker RM, Daniel KM (2012) Detection of TiO2 nanoparticles in cells by flow cytometry. In: Soloviev M (ed) Nanoparticles in biology and medicine. Methods Mol Biol, vol 906. Humana Press, Totowa, New Jersey, pp 497–509CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Reproductive and Developmental Toxicology Branch, Public Health and Integrated Toxicology Division, Center for Public Health and Environmental AssessmentOffice of Research and Development U.S. Environmental Protection AgencyResearch Triangle ParkUSA
  2. 2.Neurological and Endocrine Toxicology Branch, Public Health and Integrated Toxicology Division, Center for Public Health and Environmental AssessmentOffice of Research and Development U.S. Environmental Protection AgencyResearch Triangle ParkUSA

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