Biomedical Engineering Reference
In-Depth Information
the counting cell. At least six numerations per sample are performed to limit the risk of error. The
percentage of viability corresponds to the percentage of living cells resistant to any adverse effect
of the NPs.
19.9.1.9 Alamar Blue Reduction
Standard cytotoxicity assays that assess chemical toxicity may generate conflicting results with
some nanomaterials (e.g., carbon nanomaterials) since dye-based assay may often provide false
visibility and cytokine data [134]. The alamar blue reagent is added to the medium at a 10% concen-
tration and incubated with cells for a few hours. The alamar blue reagent is converted from the non-
fluorescent indicator dye resazurin into the highly red fluorescent metabolite resorufin via reduction
reactions by metabolically active cells [134]. The fluorescence of each well is quantified on a spec-
trophotometer with excitation and emission wavelength. The resulting fluorescence is proportional
to the number of viable cells per well. Fluorescence values are normalized to media controls and
expressed as percent viability. Experimental controls are also conducted with the alamar blue to
assess potential NPs/assay interaction [134].
19.9.1.10 Commassie Blue Assay
The cell viability of NPs is also determined by commassie blue assay [135]. In this method, the cul-
ture medium is treated with Bradford reagent. The optical density of the medium is determined at a
particular wavelength using enzyme-linked immunosorbent assay (ELISA) plate reader.
19.9.1.11 MetPLATE E. coli Bioassay
This method is used to evaluate the potential toxicity of several metal-based NPs (e.g., nAg, nTiO 2 ,
nZnO, and CdSe QDs) on E. coli . It is a β-galactosidase-based assay in which chlorophenol red
β-galactopyranoside (CRPG) is used as a chromogenic substrate that is cleaved by the enzyme,
forming galactopyranose and chlorophenol red as the by-products. During this event, yellow-
colored CRPG is transformed into magneta-colored chlorophenol red, the concentration of which
is proportional to the activity of β-galactosidase, which is quantified at a particular wavelength
using the microplate reader. Moderately hard water is used as a negative control, while Cu 2+ L −1 (as
CuSO 4 ) is used as a positive control with each set of analysis [136].
19.9.1.12 CellTiter Assay
Mitochondrial function was assessed using the CellTiter 96 AQueous one-solution assay
[137]. The reagent contains tetrazolium compound [3-(4,5-dimethylthiazole-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MST(a)] and electron-
coupling reagent (phenazine ethosulfate). The absorbance is measured at a particular wavelength
with a standard microplate reader. The quantity of formazan product as measured by the micro-
plate reader is directly proportional to the number of living cells in culture. The relative cell
viability (%) related to control containing cell culture medium without NPs is calculated by [A] test /
[A] control × 100 where [A] test is the absorbance of the test sample and [A] control is the absorbance of
the control sample [137].
19.9.1.13 Annexin V/Propidium Iodide Staining for Apoptotic and Necrotic Cells
Annexin V (VAC alpha), which is regularly used to detect apoptotic cells [138], strongly binds to
phosphatidyl serine in a calcium-dependent manner. Phosphatidyl serine is normally excluded from
the extracellular side of the plasma membrane, but flips between the inner and the outer side upon
the onset of apoptosis [138]. Fluorescently labeled Annexin V can therefore be used to detect apop-
totic cells [138]. Necrotic cells will allow Annexin V to bind the inner part of the plasma membrane,
resulting in false-negative results due to cell disintegration. Hence, cells have to be costained with
propidium iodide (3,8-diamino-5-[3-(diethylmethylammonio) propyl]-6-phenylphenanthridinium
diiodide) that will exclusively stain necrotic cells [139,140]. Investigation of apoptosis via Annexin
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