Biomedical Engineering Reference
In-Depth Information
V staining and flow cytometry has been reported, for example, for TiO
2
NPs [141], for pure and
polyhydroxylated fullerenes [142], for SWCNTs (111), and for QDs [143].
As apoptotic cells may easily detach from their substrate, it is essential to collect adherent and
floating cells for Annexin V staining [144]. Consequently, NPs in the cell culture supernatant will
be present in the cell suspension despite several washing steps. Gold NPs have been shown to bind
propidium iodide and have to be taken up by intact cell culture cells. This process leads to false-
positive results in the detection of necrotic cells [145].
19.9.1.14 Detection of the Apoptosis Marker Caspase-3
The detection of active Caspase-3 is one of the most commonly used apoptosis assays. Apoptosis
may be triggered by different elicitors activating two main signaling cascades that converge in the
activation of Caspase-3 [146]. The cysteine protease Caspase-3 is produced as a zymogen in the
cytosol and is activated in the terminal apoptotic cascade by cleavage [113]. As soon as Caspase-3
is activated, cell death is inevitable. Activated Caspase-3 can be detected by measuring the cleav-
age of a Caspase-3 substrate (preferably the amino acids DEVD) linked to a chromophore (pNA)
or fluorophore (AFC, AMC) that absorbs or emits light when separated from the substrate [114].
As yet, the Caspase-3 assay has been utilized to examine apoptosis in cell culture cells upon
exposure, for example, to fullerenes [147], SWCNTs [148], silica NPs [149], QDs [143], and to
TiO
2
NPs [64].
Caspase-3 is inhibited by trace metal ions, especially by Zn
2+
ions [150]. On the other hand,
Caspase-3 is relatively unsusceptible to changes in pH [150]. As described above, apoptotic cells
may easily detach from their substrate, so that adherent and floating cells have to be used for
Caspase-3 activity assays [144]. NPs in the cell culture supernatant will therefore remain in the cell
suspension during measurement.
19.9.2
s
tress
r
espoNse
19.9.2.1 Detection of ROS
Cellular stress response is often investigated with H
2
DCF-DA (20,70-dichlorodihydrofluoresc(e) in
diacetate), which is a widely used probe for the
in vitro
detection of intracellular ROS [151].
The acetylated nonfluorescent molecule is taken up by cell culture cells, is presumably trapped
in the cytosol by deacetylation, and becomes fluorescent upon intracellular oxidation [152]. Several
studies on the exact mechanism responsible for H
2
DCF oxidation offer varying conclusions; conse-
quently, it has been suggested that DCF should be applied as a qualitative marker for cellular oxida-
tive stress in general [152]. A possible increase in DCF fluorescence has been investigated after cell
culture exposure [152].
Internalized, deacetylated H
2
DCF does not exclusively remain in the cytosol but may accumulate
in the extracellular space and react with catalytically active substances outside the cells [153]. DCF
fluorescence is strongly pH dependent [154]. Additionally, NPs such as carbon-based materials may
absorb light so that DCF fluorescence may be quenched. Controls including NPs and oxidized DCF
have been suggested [155], but need to be verified for a linear relationship between NPs and dye
concentration versus the decrease in fluorescence.
Exposure to NPs is known to cause an increase in ROS, which could lead to oxidative stress.
ROS generation by NPs could be due to three factors [156]: (i) active redox cycling on the surface
of NPs, particularly the metal-based NPs [157]; (ii) oxidative groups functionalized on NPs; and
(iii) particle-cell interactions, especially in the lungs where there is a rich pool of ROS producers
such as the inflammatory phagocytes, neutrophils, and macrophages (Figure 19.5). Overproduction
of ROS activates a series of cytokine cascades, which include an upregulation of interleukins (ILs),
kinases, and tumor necrosis factor (TNF-α) proinflammatory signaling processes as a counterreac-
tion to oxidative stress [158]. Studies on TiO
2
NPs and C
60
fullerenes have shown that these NPs
induce elevation of proinflammatory enzymes, such as IL-1, TNF-α, IL-6, macrophage inhibitory
