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Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line

MONTEIRO-RIVIERE, Nancy A and Inman, Alfred O and Zhang, L.W. (2009) Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line. Toxicology and applied pharmacology, 234 (2). pp. 222-235. ISSN 0041-008X

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Single-walled carbon nanotubes (SWCNT), fullerenes (C60), carbon black (CB), nC60, and quantum dots (QD) have been studied in vitro to determine their toxicity in a number of cell types. Here, we report that classical dye-based assays such as MTT and neutral red (NR) that determine cell viability produce invalid results with some NM (nanomaterials) due to NM/dye interactions and/or NM adsorption of the dye/dye products. In this study, human epidermal keratinocytes (HEK) were exposed in vitro to CB, SWCNT, C60, nC60, and QD to assess viability with calcein AM (CAM), Live/Dead (LD), NR, MTT, Celltiter 96® AQueous One (96 AQ), alamar Blue (aB), Celltiter-Blue® (CTB), CytoTox One™ (CTO), and flow cytometry. In addition, trypan blue (TB) was quantitated by light microscopy. Assay linearity (R2 value) was determined with HEK plated at concentrations from 0 to 25,000 cells per well in 96-well plates. HEK were treated with serial dilutions of each NM for 24 h and assessed with each of the viability assays. TB, CAM and LD assays, which depend on direct staining of living and/or dead cells, were difficult to interpret due to physical interference of the NM with cells. Results of the dye-based assays varied a great deal, depending on the interactions of the dye/dye product with the carbon nanomaterials (CNM). Results show the optimal high throughput assay for use with carbon and noncarbon NM was 96 AQ. This study shows that, unlike small molecules, CNM interact with assay markers to cause variable results with classical toxicology assays and may not be suitable for assessing nanoparticle cytotoxicity. Therefore, more than one assay may be required when determining nanoparticle toxicity for risk assessment. Keywords: Carbon; Nanomaterials; Carbon black; Single-walled carbon nanotubes; C60; nC60; Quantum dots; Calcein AM; Live/Dead; NR; MTT; Celltiter 96® AQueous One; Alamar Blue; Celltiter-Blue®; CytoTox One™; Flow cytometry; Human epidermal keratinocytes; Toxicity; Viability assays Abbreviations: NM, nanomaterials; CNM, carbon nanomaterials; CNT, carbon nanotubes; C60, fullerenes; HEK, human epidermal keratinocytes; SWCNT, single-walled carbon nanotubes; MWCNT, multi-walled carbon nanotubes; IL-8, interleukin 8; CB, carbon black; QD, quantum dots; NR, neutral red; KGM-2, keratinocyte growth medium; TB, trypan blue exclusion assay; CAM, calcein AM; LD, Live/Dead®; 96 AQ, CellTiter 96® AQueous One; aB, alamar Blue®; CTB, Celltiter-Blue®; CTO, CytoTox One™ Homogeneous Membrane Integrity Assay; EthD-1, ethidium homodimer-1; LDH, lactate dehydrogenase; TEM, transmission electron microscopy

Item Type:Article
Subjects:NanoSafety > Environment, health and safety aspects of nanotechnology
ID Code:10658
Deposited By:M T V
Deposited On:15 Mar 2011 11:09
Last Modified:15 Mar 2011 11:09

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