Biomedical Engineering Reference
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Hillegass et al. 2010; Pujalte et al. 2011; Akhtar et al. 2012; Saquib et al. 2012) and the clonogenic
assay, based on the ability of a single cell to grow into a colony (Hillegass et al. 2010; Poonam
et al. 2011).
5.4.2.2 Direct/Indirect Intracellular ROS Measurement
The effects of ROS on cell metabolism typically involve the mechanisms in the apoptosis process.
Direct/indirect intracellular ROS measurement assays include the glutathione (GSH) assay, a lumi-
nescent-based assay for the detection and quantification of GSH in cells (Jones and Grainger 2009;
Fahmy and Cormier 2009; Pan et al. 2009; Poonam et al. 2011; Pujalte et al. 2011; Saquib et al.
2012; Akhtar et al. 2012); lipid peroxidation measurement, which measures increased concentra-
tions of end products of lipid peroxidation, indicating increased oxidative damages in the cells (Li
et al. 2008; Fahmy and Cormier 2009; Jones and Grainger 2009; Hillegass et al. 2010; Poonam et al.
2011); 2-, 7-dichlorofluorescein (DCFH) assay, which detects intracellular DCFH oxidation due to
the presence of hydrogen peroxides (Bhattacharya et  al. 2009; Jones and Grainger 2009; Fahmy
and Cormier 2009; Pan et al. 2009; Pujalte et al. 2011; Poonam et al. 2011; Akhtar et al. 2012); and
electroparamagnetic resonance (EPR) assay to directly measure free radicals in cells and tissues
(Bhattacharya et al. 2009; Poonam et al. 2011).
5.4.2.3 Assays on the Genomic Level
Assays on the genomic level measure any damage that occurs to the DNA of the cells. Examples
of this type of assay include the comet assay, also known as single-cell gel electrophoresis assay, a
technique for the detection of DNA damage at the level of the individual eukaryotic cell (Lai et al.
2008; Mroz et al. 2008; Bhattacharya et al. 2009; Jones and Grainger 2009; Hillegass et al. 2010;
Poonam et al. 2011; Mei et al. 2012; Saquib et al. 2012); and DNA damage biomarker assay, as it is
well known that excessive generation of ROS can oxidize cellular biomolecules and the resulting
free radicals also lead to oxidative modifications in DNA, including strand breaks and base oxida-
tions (Bhattacharya et al. 2009; Hillegass et al. 2010; Song et al. 2012).
5.4.3 B IoseNsINg a pproaches for I NflaMMatory B IoMarkers d etectIoN
Most current in vitro cytotoxicity assays rely on the evaluation of cell viability as discussed previ-
ously. However, the cellular stress and inflammatory responses can also be used to evaluate cellular
toxicity by the detection of specific biomarkers (Kroll et  al. 2009). Cytokines are of particular
interest as one group of such biomarkers, and their release by cells has been studied as a marker
of a cellular immune response (Pfaller et al. 2009). Cytokines regulate the growth and function of
immune cells during inflammation and in later immune responses (Pfaller et al. 2009). Therefore,
the concentration of cytokines in the medium, typically below 10 ng/mL, will increase in response
to inflammation.
Immunoassays are often used for the in vitro detection of secreted cytokines. Their principles are
based on capturing cytokines by a specific antibody and then measuring their levels. A sandwich
immunoassay format has been used for the detection of interleukin 6 (IL-6), interleukin 8 (IL-8),
or monocyte chemotactic protein-1 (MCP-1) (Ida et al. 1992; Kajikawa et al. 1996; Battaglia et al.
2005; Liu et al. 2005; Yang et al. 2005; Tan et al. 2008; Kemmler et al. 2009; Pfafflin and Schleicher
2009). In particular, enzyme-linked immunosorbent assay (ELISA) is a simple and sensitive method
for monitoring the effects of nanoparticles on immune cells by assessing the levels of cytokines that
are released into the cell culture supernatant after the addition of nanoparticles to a cell culture. The
ELISA method was first described in 1971 (Lequin 2005), and enables the simple and accurate quan-
tification of inflammatory markers in cell culture supernatants through antibodies and enzymatic
detection reactions. ELISA results have been reported for nanoparticles of different compositions
and origins, for example, for titanium dioxide (Tao and Kobzik 2002), iron oxide (Wottrich et al.
2004), zinc oxide, carbon black (Monteiro-Riviere and Inman 2006; Duffin et  al.  2007), carbon
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