Biomedical Engineering Reference
In-Depth Information
19.9.1.4 WST-1 Cytotoxicity Assay
Measurement of cellular metabolic activity by 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-3H-5-
tetrazolio]-1,3-benzene disulfonate (WST-1) assay is based on the cleavage of the tetrazolium
salt WST-1 to soluble formazan by succinate-tetrazolium reductase [120]. Succinate-tetrazolium
reductase is a mitochondrial enzyme that is active only in viable cells. The amount of formazan is
proportional to the mitochondrial activity. There are various ways in which the NPs may interfere
with the WST-1 assay. Therefore, two different WST-1 interference tests are performed. The first
interference test is designed to whether NPs (1) scatter or absorb light or (2) interference with the
WST-1 reagent.
19.9.1.5 Clonogenic Assay
The clonogenic assay determines the ability of cells to form colonies after toxic treatment. Contrary
to the WST-1 assay, it has the advantage of not using a colorimetric indicator dye. In fact, nanoma-
terials are known to frequently interact with the colorimetric indicator used in cytotoxicity assays
[121]. Clonogenic assay has been applied to assess the cytotoxicity potential of various NPs, for
instance, SWCNT [122], silica NPs [121], and so on.
19.9.1.6 Hematoxylin and Eosin Assay
In this method, the cells are treated with different concentrations of NPs and fixed with 4% polyoxy-
methylene. Then the cells are stained with hematoxylin and eosin and observed under a microscope.
In general, the eosin imparts a pink-to-red color of the cytoplasm, and hematoxylin stains nucleus
blue. This method has been applied for the assessment of cytotoxicity potential of silica NPs [123].
19.9.1.7 Neutral Red Uptake Assay
Since 1894, neutral red has been used as a viability stain and has since been implemented in numer-
ous cytotoxicity, cell proliferation, and adhesion assays [124]. Neutral red (3-amino-7-dimethyl-
amino-2-methylphenazine hydrochloride) is weakly cationic, and is thought to be taken up into the
cytosol by nonionic diffusion through the cell membrane to then accumulate in the lysosomes of
viable cells, while it is excluded from dead cells [125]. The uptake of neutral red may be detected
via fluorescence or absorption measurement. So far, the neutral red uptake (NRU) in NIH3T3
3
T
3
mouse
fibroblasts is the only validated
in vitro
method for toxicity testing [126]. A number of different
NPs such as TiO
2
[127], SWCNTs and MWCNTs [128], and chitosan NPs [129] have been tested
by NRU assays.
The color and intensity of light emission of neutral red is pH dependent [130]. It is known that
neutral red has a high affinity to lipophilic structures (such as suberin or phenolic substances), the
protonated dye interacts with negative charges, and covalently binds to cellular structures [130].
These properties were utilized to study the adsorption of neutral red to mercaptoethane sulfonate-
protected gold NPs. The optical properties of neutral red were significantly influenced, and the NPs
exerted static and dynamic energy transfer quenching of neutral red fluorescence [131]. Furthermore,
SWCNTs have been shown to interact with neutral red and deplete the dye from the cell supernatant
leading to false-positive results [132]. Hence, properties that are useful for technical applications
may be deleterious in cytotoxicity testing.
19.9.1.8 Trypan Blue Exclusion Assay
Trypan blue is one of the many dye-recommended techniques of exclusion staining, counting and
evaluation of cellular population, and acute cellular toxicity [133]. This method is based on the
principle that the living cells do not incorporate the dye, whereas dead cells incorporate it owing
to their damaged membrane. Cells are treated with different concentrations of NPs in well plates
and after 24 h incubation, cells are trypsinized. Unexposed control cultures are maintained under
the same conditions. The enumeration is carried out by immediate microscopic observation using
