Abstract
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 was used to measure changes in light scatter from cells after incubation with TiO2 nanoparticle. Both the side scatter and forward scatter changed substantially in response to the TiO2. Between 0.1 and 30 μg/mL TiO2, the side scatter increased sequentially while the forward scatter decreased, presumably due to substantial light reflection by the TiO2 particles. At the lowest concentrations of TiO2 (0.1–0.3 μg/mL), the flow cytometer apparently could detect as few as 5–10 nanoparticles per cell as shown using dark field microscopy. The influence of nanoparticles on the cell cycle was detected by nonionic detergent lysis of nanoparticle-incubated cells. Viability of nanoparticle-treated cells was determined by PI exclusion.
These data suggest that the uptake of nanoparticles within cells can be monitored using flow cytometry and confirmed by dark field microscopy. This approach may help fill a critical need to assess the relationship between nanoparticle dose and cellular toxicity. Such experiments could potentially be performed quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.
Contractor to the USEPA, award # EP09D000042.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Maynard AD, Aitken RJ, Butz T et al (2006) Safe handeling of nanotechnology. Nature 444:267–269
Auffan M, Rose J, Bottero JY et al (2009) Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. Nat Nanotechnol 4(10):634–641
Nohynek GJ, Lademann J, Ribaud C et al (2007) Grey goo on the skin? Nanotechnology, cosmetic and sunscreen safety. Crit Rev Toxicol 37(3):251–277
Salzman GC (2001) Light scatter: detection and usage. Curr Protoc Cytom Chapter 1:Unit 1.13
Shapiro HM (2003) Practical flow cytometry, 3rd edn. Wiley, Hoboken, NJ
Shapiro HM (2001) Optical measurements in cytometry: light scattering, extinction, absorption, and fluorescence. Methods Cell Biol 63:107–129
Steen HB (2004) Flow cytometer for measurement of the light scattering of viral and other submicroscopic particles. Cytometry A 57(2):94–99
Zucker RM, Elstein KH, Easterling RE et al (1988) Flow cytometric discrimination of nuclei by right angle scatter. Cytometry 9:226–231
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 10:312–319
Zucker RM, Perrault SD, Elstein KH (1992) Utility of light scatter in the morphological analysis of sperm. Cytometry 13:39–47
Giaretti W, Nüsse M (1994) Light scatter of isolated cell nuclei as a parameter discriminating the cell-cycle subcompartments. Methods Cell Biol 41:389–400
Zucker RM, Massaro EJ, Sanders KM et al (2010) Detection of TiO2 nanoparticles in cells by flow cytometry. Cytometry A 77(7):677–685
Stringer B, Imrich A, Kobzik L (1995) Flow cytometric assay of lung macrophage uptake of environmental particulates. Cytometry 20(1):23–32
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(8):3018–3024
Zucker RM (2008) Flow cytometry quality assurance. Standardization and quality assurance in fluorescence measurements 2. Springer series on fluorescence. In: Resch-Genger U (volume editor), Wolfbeis OS (series editor) Methods and applications, Springer, Berlin, pp 343–370
Tanev S, Sun W, Pond J (2009) Flow cytometry with gold nanoparticles and their clusters as scattering contrast agents: FDTD simulation of light-cell interaction. J Biophotonics 2(8–9):505–520
Jain PK, Lee KS, El-Sayed IH (2006) Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition: applications in biological imaging and biomedicine. J Phys Chem B 110(14):7238–7248
Bohmer RM, King NJ (1984) Flow cytometric analysis of immunogold cell surface. Cytometry 5:543–546
Acknowledgements
Thanks are extended to Carl Blackman, Will Boyes, Laura Degn, Sarah Hutchinson, and Kristen Sanders for their helpful comments.
Although the research described in this article has been supported by the United States Environmental Protection Agency, it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zucker, R.M., Daniel, K.M. (2012). Detection of TiO2 Nanoparticles in Cells by Flow Cytometry. In: Soloviev, M. (eds) Nanoparticles in Biology and Medicine. Methods in Molecular Biology, vol 906. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-953-2_40
Download citation
DOI: https://doi.org/10.1007/978-1-61779-953-2_40
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-952-5
Online ISBN: 978-1-61779-953-2
eBook Packages: Springer Protocols