A006 2

Manufactured by Nanjing Jiancheng

Sourced in China

The A006-2 is a laboratory equipment product. It is designed for specific laboratory functions. No further details about its core function or intended use can be provided in an unbiased and factual manner.

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17 protocols using a006 2

Quantifying Cellular Glutathione Levels

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The relative GSH concentration in cell lysates was assessed using a kit from Nanjing Jiancheng (#A006-2) according to the manufacturer’s instructions. The measurement of GSH used a kinetic assay, in which catalytic amounts (nmoles) of GSH caused a continuous reduction of 5, 5′-dithiobis (2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid and the GSSG formed was recycled by glutathione reductase and NADPH. The yellow product (5-thio-2-nitrobenzoic acid) was measured spectrophotometrically at 420 nm. The values were normalized by protein concentration and presented as percentages relative to the negative control.

Cao J., Chen X., Jiang L., Lu B., Yuan M., Zhu D., Zhu H., He Q., Yang B, & Ying M. (2020). DJ-1 suppresses ferroptosis through preserving the activity of S-adenosyl homocysteine hydrolase. Nature Communications, 11, 1251.

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Antioxidant and Lipid Biomarkers in Liver

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The levels of SOD, GSH, MDA and triglyceride in liver tissue homogenates from each group were measured using the corresponding kits (Catalog# A001-3, A006-2, A003-1, and A110-1, respectively) from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocols. CYP2E1 activity was assayed by the rate of oxidation of p-nitrophenol to p-nitrocatechol according to a previously described protocol.^{68 (link)}

Zhong F., Hu Z., Jiang K., Lei B., Wu Z., Yuan G., Luo H., Dong C., Tang B., Zheng C., Yang S., Zeng Y., Guo Z., Yu S., Su H., Zhang G., Qiu X., Tomlinson S, & He S. (2019). Complement C3 activation regulates the production of tRNA-derived fragments Gly-tRFs and promotes alcohol-induced liver injury and steatosis. Cell Research, 29(7), 548-561.

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Oxidative Stress Biomarkers Quantification

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After determining the concentration of total protein in the supernatant of the pulmonary homogenate by the bicinchoninic acid method, the GSH and MDA contents and GSH-Px, (SOD), and CAT activities were measured by biochemical method following the instruction of reagent kits (A006-2, A003-1, A005, A001-3 and A007-1, Nanjing Jiancheng Bioengineering Institute, China). The GSH assay was based on the development of a yellow color when 5,5′-dithio-bis-nitrobenzoic acid was added to compounds containing sulfhydryl groups. The MDA assay was determined by the thiobarbituric acid colorimetric method. The GSH-Px activity was detected by the consumption of GSH. The SOD activity was determined by the xanthine oxidase method. The CAT activity was determined by the H₂O₂ decomposition rate. The values were expressed as nmol/mg protein for GSH and MDA and units (U)/mg or U/g of protein for GSH-Px, SOD, and CAT.

Wang F., Zuo Z., Chen K., Fang J., Cui H., Shu G., Zhou Y., Chen Z., Huang C, & Liu W. (2018). Histopathological Changes Caused by Inflammation and Oxidative Stress in Diet-Induced-Obese Mouse following Experimental Lung Injury. Scientific Reports, 8, 14250.

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Colonic Oxidative Stress Biomarkers

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Proteins from homogenized colonic tissues were quantified using a BCA protein assay kit (P0009, Beyotime, Shanghai, China) and prepared to determine the concentration of malondialdehyde (MDA; A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and glutathione (GSH; A006-2, Nanjing Jiancheng Bioengineering Institute), as well as the activity of superoxide dismutase (SOD; A001-1, Nanjing Jiancheng Bioengineering Institute).

Han J., Li W., Shi G., Huang Y., Sun X., Sun N, & Jiang D. (2022). Atractylenolide III Improves Mitochondrial Function and Protects Against Ulcerative Colitis by Activating AMPK/SIRT1/PGC-1α. Mediators of Inflammation, 2022, 9129984.

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Glutathione Levels in Spinal Cord Injury

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Glutathione (GSH) concentrations in the T10 spinal cord specimens were measured on days 1, 3, and 7 post-injury using a GSH assay kit (A006-2, Nanjing Jiancheng Bioengineering Institute). Briefly, supernatant (0.1 mL) from the spinal cord homogenate was added to 0.1 mL of reagent 1, and the samples were centrifuged for 10 minutes at 986 × g. After centrifugation, the supernatants were collected. Sample detection was performed in accordance with the kit instructions. After the color reaction, the absorbance values at 405 nm were measured using a microplate reader (BioTek Epoch, Winooski, VT, USA). The absorbance values for the blank, standard, and experimental wells were recorded, and the experiments were repeated three times. The GSH concentrations were calculated according to the following formula: GSH concentration = (measured OD value – blank OD value)/(standard OD value – blank OD value) × GSH standard concentration (20 μM) × dilution factor /protein concentration of homogenate.

Chen Y.X., Zuliyaer T., Liu B., Guo S., Yang D.G., Gao F., Yu Y., Yang M.L., Du L.J, & Li J.J. (2022). Sodium selenite promotes neurological function recovery after spinal cord injury by inhibiting ferroptosis. Neural Regeneration Research, 17(12), 2702-2709.

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Oxidative Stress Biomarker Assessment in Serum

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Whole blood samples were collected from the eye socket using 10% chloral hydrate and centrifuged at 1,000 × g for 20 min at 4°C to collect serum. After cooling to room temperature, 150 µl serum was used to detect the activities of glutathione peroxidase (GSH-Px; A005), glutathione (GSH; A006-2), superoxide dismutase (SOD; A001-3) and malondialdehyde (MDA; A003-1; all Nanjing Jiancheng Biology Engineering Institute) using ELISA kits and a spectrophotometer (GE Healthcare Life Sciences) according to the manufacturer's instructions.

Ding Y., Wang L., Song J, & Zhou S. (2017). Protective effects of ellagic acid against tetrachloride-induced cirrhosis in mice through the inhibition of reactive oxygen species formation and angiogenesis. Experimental and Therapeutic Medicine, 14(4), 3375-3380.

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Colorimetric Assay of Liver Glutathione

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Liver GSH was measured using colorimetric kits (#A006-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The tissue was homogenized in mataphosphoric acid, and centrifuged at 3000g at 4°C for 10min. The upper aqueous layer was collected for measurement and mixed with 20μL sample and 120μL DTNB: Glutathian Reductase=1:1 solution; after 30s, 60μL B-NADPH was added and the absorbance at 412 nm was immediately read in microplate reader. The rate of 2-nitro-5-thiobenzoic acid formation was then calculated from a standard curve.

Huo Y., Yin S., Yan M., Win S., Than T.A., Aghajan M., Hu H, & Kaplowitz N. (2017). Protective role of p53 in acetaminophen hepatotoxicity. Free radical biology & medicine, 106, 111-117.

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Relative GSH Concentration Assay

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The cell lysate’s relative GSH concentration was measured using an assay kit from Nanjing Jiancheng (#A006-2) according to the manufacturer’s instructions. The luminescence of yellow product of the reaction was measured at 405 nm and normalized by protein concentration.

Hu X., He Y., Han Z., Liu W., Liu D., Zhang X., Chen L., Qi L., Chen L., Luo Y., Li Q., Chen P., Wu Q., Zhu X, & Guo H. (2022). PNO1 inhibits autophagy-mediated ferroptosis by GSH metabolic reprogramming in hepatocellular carcinoma. Cell Death & Disease, 13(11), 1010.

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Quantifying Cellular Redox Homeostasis

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Protein samples were yielded from tissues and cells to determine GSH levels using a reduced GSH detection kit (A006-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). Following the producer’s instructions, reactive oxygen species (ROS) levels in tissues and cells were examined using the fluorescent probe DCFH-DA (S0033S, Beyotime, Shanghai, China). The lysed tissues and cells were added with DCFH-DA and incubated at 37 ^∘C for 20 min and washed with PBS 3 times. The changes in the fluorescent intensity at a wavelength of 500/530 nm were determined using a fluorescent microplate reader (Bio-Rad, Hercules, CA, USA), and the results were compared to the fluorescence intensity of the control group.

Chen Z., Gu Q, & Chen R. (2023). Promotive role of IRF7 in ferroptosis of colonic epithelial cells in ulcerative colitis by the miR-375-3p/SLC11A2 axis. Biomolecules and Biomedicine, 23(3), 437-449.

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Intracellular Glutathione Quantification

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The experimental grouping and treatment were conducted as described in method 2.4. After incubation, cells were made into cell suspensions by trypsin digestion and washed with PBS three times, followed by homogenate using an ultrasonic cell disruption system (JY92-IIN, Xinzhi, Ningbo, China). Then the intracellular glutathione was measured using a glutathione determination kit (A006-2, Jiancheng, Nanjing, China) in accordance with the supplier's instructions. The intracellular glutathione was evaluated based on the absorbance at 420 nm using a fluorescence microplate reader (SpectraMax M5, MD, CA, USA), with excitation wavelength at 350 nm.

Wang X., Cui Y.J., Qi J., Zhu M.M., Zhang T.L., Cheng M., Liu S.M, & Wang G.C. (2018). Fucoxanthin Exerts Cytoprotective Effects against Hydrogen Peroxide-induced Oxidative Damage in L02 Cells. BioMed Research International, 2018, 1085073.

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