A portion of the hippocampus was reserved for the determination of oxidized glutathione (GSSG) and reduced glutathione (GSH) levels using the GSH/GSSG Ratio Detection Assay Kit II (Abcam, Boston, MA, USA), following the manufacturer’s instructions. Briefly, tissue samples were homogenized in ice-cold PBS/0.5% NP-40 with a mini Potter–Elvehjem pestle ((Sigma-Aldrich, St. Louis, MO, USA).), centrifuged for cell debris removal, and deproteinized to remove enzymes that could potentially metabolize glutathione (Deproteinizing Sample Preparation Kit—TCA, Abcam, Boston, MA, USA).
Gsh gssg ratio detection assay kit 2
Manufactured by Abcam
Sourced in United Kingdom, United States
The GSH/GSSG Ratio Detection Assay Kit II is a colorimetric assay designed to measure the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in biological samples. The kit provides a simple and efficient method to quantify this important redox pair, which is essential for evaluating cellular oxidative stress levels.
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Lab products found in correlation
Deproteinizing sample preparation kit tca, by Abcam (2 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Iron assay kit, by Abcam (1 mentions)
Ab118970, by Abcam (1 mentions)
Sta 330, by Cell Biolabs (1 mentions)
Oxiselect in vitro ros rns assay kit, by Cell Biolabs (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Flx800, by Agilent Technologies (1 mentions)
Mda lipid peroxidation microplate assay, by Merck Group (1 mentions)
Mammalian cell lysis buffer, by Abcam (1 mentions)
Nadp nadph glo assay, by Promega (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Gsh and gssg assay kit, by Beyotime (1 mentions)
Beadbug microtube homogenizer, by Benchmark Scientific (1 mentions)
3 mm zirconium beads, by Benchmark Scientific (1 mentions)
Luminescent atp detection kit, by Abcam (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
17 protocols using gsh gssg ratio detection assay kit 2
1
Glutathione Redox Status Quantification
2
Evaluating Ferroptosis Mechanisms
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Various cell biological methods were used to evaluate ferroptosis. Lipid peroxidation was assessed using the BODIPYTM 581/591 C11 (D3861, Thermo Fisher Scientific), and oxidation of the polyunsaturated butadienyl portion of the dye results in the shifting of the fluorescence emission peak from ∼590 (red) to ∼510 nm (green). PWR1E or DU145 cell lines were incubated with 10 μM erastin for 24 h. Then 1 μg/ml of Hoechst 33342 and 1 μm of BODIPYTM 581/591 C11 were added to the culture medium for living cell imaging at the last hour of incubation. Ferroptosis-related indexes, including the levels of Fe2+ release, malondialdehyde (MDA), reactive oxygen species (ROS), and GSH, were determined using the Iron Assay Kit (ab83366, Abcam, United Kingdom), lipid peroxidation (MDA) assay (ab118970, Abcam), DCF ROS/RNS Assay (ab238535, Abcam), and GSH/GSSG Ratio Detection Assay Kit II (ab205811, Abcam), respectively.
3
Glutathione Redox Status Quantification
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GSH/GSSG Ratio Detection Assay Kit II (Abcam, ab205811) was used to detect reduced (GSH) and oxidized (GSSG) glutathione levels. Cells were seeded in a 12‐well plate and after completion of the experiment, medium was removed and cells were washed with cold PBS. Cells were lysed, homogenized for 15 min, and clear supernatant was collected and transferred to a new tube. For GSH detection, 50 µl of GSH assay mixture was added to standards and samples. For GSSG measurement, 50 µl of total glutathione assay mixture was added to standards and samples. After incubation of 15 min, fluorescence was measured at Ex/Em = 490/520 nm with fluorescence microplate reader.
4
Hepatic Lipid and Oxidative Assessment
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Liver triglyceride (TriG) content was extracted by homogenization of each liver tissue sample with a mixture of 1:2 chloroform and methanol (v/v) as described previously (Shen et al., 2017 (link)). The hepatic content of TriG was determined using a TriG assay kit (TR0100, Sigma). Hepatic levels of lipid peroxidation products (malondialdehyde, MDA) were determined by thiobarbituric acid reactive substances (TBARS) assay using a commercially available kit (STA‐330, Cell Biolabs). Quantification of total reactive oxygen species (ROS) and reactive nitrogen species (RNS) present in the liver tissue was carried out using a specific ROS/RNS probe, namely dichlorodihydrofluorescein DiOxyQ (DCFH‐DiOxyQ), through the use of a OxiSelect in vitro ROS/RNS assay kit (STA‐347, Cell Biolabs). The levels were normalized to total protein amount of liver tissues. The levels of GSH and GSSG were measured using a GSH/GSSG Ratio Detection Assay Kit II (ab205811, Abcam).
5
Oxidative Stress Evaluation in Ovarian Cancer
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To evaluate lipid peroxidation or cellular GSH/GSSG in OvCa cells, the cells were treated with indicated treatments for 48 h. For confocal imaging, cell lipid peroxidation was detected by staining the fluorescent dye C11-BODIPY581/591 (Thermo Fisher) with the probe (2.5 µM) for 30 mins at 37 °C. Mounting media was used as an anti-fade reagent. Images were acquired using inverted microscopy (Carl Zeiss LSM 880). For flow cytometry, the cells were washed subsequent to staining with C11-BODIPY and analyzed by a BD LSRII flow cytometer (Becton Dickinson). The cellular GSH/GSSG ratios in OvCa cells were detected using the GSH/GSSG Ratio Detection Assay Kit II (Abcam, Fluorometric-Green) according to the manufacturer's instructions.
6
Quantification of Glutathione Redox State
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Cells were re-dispensed in ice-cold lysis buffer (0.5% nonyl phenoxypolyethoxylethanol in PBS). Samples were deproteinized by trichloroacetic acid and neutralized by sodium hydrogen carbonate. GSH/GSSG ratio was measured using the GSH/GSSG ratio detection assay kit II (Abcam, Cambridge, UK) following the manufacturer’s instructions. Upon reacting with GSH, the non-fluorescent green dye used in this assay becomes fluorescent. Total and reduced GSH levels were measured with 485/20 nm excitation and 528/20 nm emission wavelengths (FLx800, Bio-Tek). Absolute GSH and GSSG levels were quantified using standard curves of GSH and GSSG.
7
Quantifying Intracellular Glutathione Redox State
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Intracellular reduced form gluthione (GSH) and its oxidized form (GSSG) were assessed using a GSH/GSSG Ratio Detection Assay Kit II (Abcam) according to the manufacturer’s instructions. Briefly, 50 μl of GSH Assay Mixture (for GSH) or Total GSH Assay Mixture (for total gluthione) solution was added to 50 μl of cell lysate sample in a 96-well plate and then incubated from light for 30 min. The output was measured on a fluorescence microplate reader at Ex/Em = 490/520 nm and concentrations of GSH and total gluthione were calculated using corresponding standard curves. GSSG concentration was calculated as following: GSSG = (total gluthione - GSH)/2.
8
Quantifying Glutathione in B Cells
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Total glutathione concentrations were measured in B lymphocytes isolated from the blood of healthy donors using GSH/GSSG Ratio Detection Assay Kit II (Abcam #205811), according to the manufacturers’ protocol.
9
Lipid Peroxidation and Glutathione Assays
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To assay lipid peroxidation, the MDA lipid peroxidation microplate assay (Merck KGaA) was used according to manufacturers' instructions. Briefly, cells were homogenized in lysis solution containing butylated hydroxytoluene. The insoluble fraction was removed by centrifugation, and the supernatant was used for analysis. The supernatants were mixed with thiobarbituric acid solution reconstituted in glacial acetic acid and then incubated at 95 °C for 60 min. The supernatants containing MDA-thiobarbituric acid adduct were added into a 96-well microplate for analysis. A microplate reader was used to measure the absorbance at OD 532 nm.
For glutathione determination, intracellular reduced form glutathione (GSH) and its oxidized form (GSSG) were assessed using a GSH/GSSG Ratio Detection Assay Kit II (Abcam, Cambridge, UK) according to the manufacturer's instructions. Briefly, cells were homogenized in 5% 5-sulfosalicylic acid, and the insoluble fraction was removed by centrifugation. The resultant supernatant was added to double-deionized H2O (ddH2O) to reduce the 5-sulfosalicylic acid concentration to 0.5% for the assay. A microplate reader was used to measure absorbance at OD 415 nm. The concentration of GSH was calculated by subtracting 2 × GSSG from the total glutathione concentration.
For glutathione determination, intracellular reduced form glutathione (GSH) and its oxidized form (GSSG) were assessed using a GSH/GSSG Ratio Detection Assay Kit II (Abcam, Cambridge, UK) according to the manufacturer's instructions. Briefly, cells were homogenized in 5% 5-sulfosalicylic acid, and the insoluble fraction was removed by centrifugation. The resultant supernatant was added to double-deionized H2O (ddH2O) to reduce the 5-sulfosalicylic acid concentration to 0.5% for the assay. A microplate reader was used to measure absorbance at OD 415 nm. The concentration of GSH was calculated by subtracting 2 × GSSG from the total glutathione concentration.
10
Quantifying Glutathione Levels
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The concentrations of GSH and total glutathione were measured using a GSH/GSSG Ratio Detection Assay Kit II; (#ab205811, Abcam Plc., Cambridge, UK). The concentration of GSSG was calculated by subtracting GSH from the total and dividing by 2. FLUOROSKAN (Thermo Fisher Scientific Inc., Waltham, MA) was used for measuring fluorescence.
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