Intracellular reactive oxygen species (ROS) was detected using an oxidant-sensitive fluorescent probe (DCFH-DA, Beyotime, Nantong, China). In brief, cells were washed twice with PBS. Then, cells were stained with 10 μM DCFH-DA at 37°C for 20 min according to the manufacturer’s instruction. DCFH-DA was deacetylated intracellularly by non-specific esterase, which was further oxidized by ROS to the fluorescent compound 2,7-dichlorofluorescein (DCF). DCF fluorescence was detected with a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, USA). To determine the oxidative status, intracellular GSH activity was measured using the GSH/GSSG Ratio Detection Assay kit (Abcam).
Gsh gssg ratio detection assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The GSH/GSSG Ratio Detection Assay Kit is a laboratory equipment product that measures the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in biological samples. This kit provides a simple and accurate method to determine the cellular redox state, which is an important indicator of oxidative stress.
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77 protocols using gsh gssg ratio detection assay kit
1
Quantifying Intracellular Oxidative Stress
2
Quantifying Glutathione Redox Homeostasis
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Measurements of glutathione and glutathione disulfide were made using the GSH/GSSG Ratio Detection Assay Kit from Abcam (ab138881), according to manufacturer's instructions. Briefly, 20 whole female heads per sample were flash frozen at 6 days (young) or 42 days (old). Samples were resuspended in 150 μl PBS with 0.5% Triton X‐100 and dissociated with a motorized pestle. Debris was removed by centrifugation and deproteinated with trichloroacetic acid (25 μl/sample) and sodium bicarbonate (80 μl 7% w/v). Samples were diluted 4× for the GSH assay, and 16× for GSSG. All samples were kept on ice using the enzymatic reaction step. The reaction mixture was incubated 45 min, then read with a BioTek fluorescence plate reader.
3
Cellular Oxidative Stress Measurement
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2 × 105 cells with stable expression of indicated genes and/or shRNAs were seeded in a well of 6-well plate, and incubated with fresh medium overnight. Cells were treated with 12 Gy ionizing radiation, and cellular oxidative stress was measured by the following methods 3 h after irradiation. The levels of GSH/GSSG ratio were determined using the GSH/GSSG Ratio Detection Assay Kit obtained from Abcam (ab138881) and quantified using a microplate reader (Ex/Em = 490/520 nm). The level of cellular DCFDA signal was measured by using Reactive Oxygen Species (ROS) Detection Assay Kit obtained from BioVision (K936-100) and evaluated by using a microplate reader (Ex/Em = 495/529 nm). The level of 8-OHdG was examined by using 8-hydroxy 2 deoxyguanosine ELISA Kit obtained from Abcam (ab201734). The data was normalized to the cell numbers.
4
Fluorometric GSH/GSSG Ratio Assay
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GSH/GSSG ratio in various U87MG cell lines was determined using GSH/GSSG Ratio Detection Assay Kit (Fluorometric-Green) (#ab138881, Abcam), according to the manufacturer's guidelines. In brief, the whole cell lysates were diluted to 1 : 80 for GSH analysis, serial dilution of GSH and GSSG stock standards were prepared as standards. A one-step fluorimetric reaction of samples with respective assay buffer and probes were incubated for 30 min. Then, fluorescence intensity was monitored at EX/EM of 490/520 nm.
5
Quantifying Striatal Glutathione Levels
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Cellular levels of glutathione (reduced/oxidized forms, GSH/GSSG) were detected from the cytosolic fraction of the striatum using a GSH/GSSG ratio detection assay kit (Abcam) according to the manufacturer’s protocol. Total 10 μg of cytosolic protein samples were loaded for the reaction with the Thiol Green Indicator Reaction Mixture and then incubated at room temperature for 30 min. Enzyme activity was detected at 490/520 nm and normalized to protein amount.
6
Glutathione Concentration Assay
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Total (GS), oxidized (GSSG), and reduced (GSH) glutathione concentrations were measured using the GSH/GSSG ratio detection assay kit (Abcam, Inc., MA, USA) and a fluorescence microplate reader with excitation and emission wavelengths of 490 and 520 nm, respectively. Each experiment was performed in triplicate.
7
Quantifying Reduced Glutathione Levels
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Cells with or without G6PD silencing were grown in 60-mm dishes. After 5 μM DOX treatment for different time periods, cells were collected and lysed in PBS with 0.5% NP-40. The reduced GSH level was determined using the GSH/GSSG ratio detection assay kit (ab138881, Abcam, USA). The excitation and emission wavelengths of the fluorescence were set at 490 nm and 520 nm, respectively, and their intensity was determined by using a spectrofluorometer (SpectraMax M5, Molecular Devices).
8
Evaluation of Hitrechol's Antioxidant and Anti-Inflammatory Effects
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To evaluate the anti-oxidative effect of Hitrechol®, catalase (CAT) activities, and superoxide dismutase (SOD) and reduced glutathione (GSH) levels were detected in collect serum and prepared liver homogenate from Groups 1 to 5 using Catalase Activity Assay Kit (ab83464), Superoxide Dismutase Activity Assay kit (ab65354) and GSH/GSSG Ratio Detection Assay kit (ab138881) according to the instructions of the manufacturer (Abcam, USA). Furthermore, to evaluate the anti-inflammatory effect of Hitrechol®, the concentration of TNF-alpha in serum were detected using ELISA kit (Abcam, USA).
9
Glutathione Redox Status Assay
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GSH and oxidized glutathione (GSSG) detection were achieved using the GSH/GSSG Ratio Detection Assay Kit (Abcam, VIC, Australia) according to the specification. GSH and GSSG were labeled with 1 × Thiol Green solution and 1 × GSSG Probe solution, respectively, and were detected by the Infinite® M200 PRO microplate reader at excitation/emission of 490/520 nm.
10
Measuring Intracellular ROS and GSH
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An oxidant-sensitive fluorescent probe (DCFH-DA) was used to detect intracellular ROS levels (Sigma-Aldrich, St. Louis, MO, USA). Briefly, the cells were washed twice by phosphate-buffered saline (PBS), and were then stained with DCFH-DA (10 μmol/l) at 37°C for 20 min as per the manufacturer’s instructions. DCFH-DA was deacetylated by intracellular non-specific esterase, and was then oxidized by ROS to generate the fluorescent compound, 2,7-dichlorofluorescein (DCF). The DCF fluorescence intensity was detected using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). Intracellular GSH activity that reflects the oxidative status of cells was determined using the GSH/GSSG Ratio Detection Assay kit (ab138881; Abcam, Cambridge, MA, USA).
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