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Preclinical Characterization of Engineered Nanoparticles Intended for Cancer Therapeutics
Published in Mansoor M. Amiji, Nanotechnology for Cancer Therapy, 2006
Anil K. Patri, Marina A. Dobrovolskaia, Stephan T. Stern, Scott E. McNeil
Biomarkers of nanoparticle-induced oxidative stress measured in our laboratory include ROS, lipid peroxidation products, and GSH/GSSG ratio. The fluorescent dichlorodihydroflourescein (DCFH) assay is used for measurement of ROS, such as hydrogen peroxide.106 DCFH-DA is a ROS probe that undergoes intracellular deacetylation, followed by ROS-mediated oxidation to a fluorescent species, with excitation 485 nm and emission 530 nm. DCFH-DA can be used to measure ROS generation in the cytoplasm and cellular organelles, such as the mitochondria. The thiobarbituric acid reactive substances (TBARS) assay is used for measurement of lipid peroxidation products, such as lipid hydroperoxides and aldehydes. A molondialdehyde (MDA) standard curve is used for quantitation. MDA, a lipid peroxidation product, combines with thiobarbituric acid in a 1:2 ratio to form a fluorescent adduct, that is measured at 521 nm (excitation) and 552 nm (emission). TBARS are expressed as MDA equivalents.107 The dithionitrobenzene (DTNB) assay is used for evaluation of glutathione homeostasis. In the DTNB assay, reduced GSH interacts with 5,5′ -dithiobis(2-nitrobenzoic acid) (DTNB) to form the colored product 2-nitro-5-thiobenzoic acid, which is measured at 415 nm, and GSSG. GSSG is then reduced by glutathione reductase to form reduced GSH, which is again measured by the preceding method. Pretreatment with thiol-masking reagent, 1-methyl-4-vinyl-pyridinium trifluoromethane sulfonate, prevents GSH measurement, resulting in measurement of GSSG alone.108
Visfatin gene expression and oxidative stress in pregnancy induced hypertension
Published in Egyptian Journal of Basic and Applied Sciences, 2018
Hanan M.A. El-Taweel, Nevein A. Salah, Amal K. Selem, A.A. El-Refaeey, A.F. Abdel-Aziz
Blood reduced glutathione content was determined by the method of Beutler et al. [23] by using a commercially available kit (Biodiagnostic, Dokki, Giza, Egypt). 0.5 ml of reagent 1 (trichloroacetic acid) was added to 0.1 ml of blood sample, centrifuged. 1.0 ml of reagent 2 (buffer) was added to 0.5 ml of the supernatant then add 0.1 ml of reagent 3 (5,5′-dithiobis(2-nitrobenzoic acid)). The color was measured at 405 nm. Thiobarbituric acid (TBA) test is widely used assay for the measurement of lipid peroxidation (malondialdehyde) according the method of Draper and Hadly, [24]. 0.5 ml serum was mixed with 2.5 ml of TCA to precipitate proteins. After centrifugation 1.0 ml of TBA was added to 2.0 ml of the supernatant and a pink chromogen was measured at 532 nm.
Bystander effect of ultraviolet A radiation protects A375 melanoma cells by induction of antioxidant defense
Published in Journal of Environmental Science and Health, Part C, 2022
One of the most popular assays to determine lipid peroxidation in cells is the thiobarbituric acid (TBA) test of MDA formed.33 The method followed was as described earlier.16 Immediately after treatments, cells were trypsinized and counted after suspending in PBS. Cold trichloroacetic acid (2 ml, 10% w⁄v) was mixed with 2 × 106 cells in 1 mL and centrifuged (3500 × g, 10 min). An equal volume of TBA (0.67% w⁄v) was added to the supernatant and boiled in a water bath for 1 h. The absorbance at 535 nm was measured to get the amount of MDA formed, expressed in nM/h/106 cells at 37° C. The molar extinction coefficient used was 1.56 × 105 M−1 cm−1.
Integrated response of the toxicity of environmentally relevant concentrations of copper in the backwater clam Meretrix casta
Published in Chemistry and Ecology, 2020
Avelyno H. D’Costa, Swizzle Furtado, S. S. Greeshma, S. K. Shyama
The malondialdehyde (MDA) assay which is used to test lipid peroxidation in the soft tissues of bivalves was carried out using a commercial kit (North West Life Science Specialities- NWK-MDA01). The assay is based on the reaction of malondialdehyde with thiobarbituric acid (TBA) forming a pink MDA-TBA2 adduct that absorbs strongly at 532 nm. To minimize the oxidation of lipids, butylated hydroxytoluene (BHT) and EDTA were added to the reaction mixture containing the sample homogenate. The activity of MDA was expressed as nmol MDA1min-1mg protein.