Cells were cultured in the 6-well plate. When the culture reaches 70–80% confluency, cells were treated with L-BSO (20 μM) for 12 hours, or treated with LA (20 mM), hydrogen peroxide (100 μM), PEITC (10 μM), PL (10 μM), Dox (10 μg/ml), or ATO (10 μM) for 4 hours respectively. Cells were rinsed twice with 2 ml ice-cold Ca2+-/Mg2+-free PBS. Cells were collected by tripsinization and lysed in 0.2 ml of ice-cold extraction buffer (0.1% Triton-X and 0.6% sulfosalicylic acid in KPE) followed by 4 cycles of freezing and thawing (1 minute in liquid nitrogen and 2 min in water bath at 37°C). Cell lysate was centrifuged and supernatant was collected for GSH and GSSG measurement according to the method described by Rahman et al.53 (link). The GSH assay is based on the chemical conjugation of GSH with 5,5′-Dithiobis(2-nitrobenzoic acid)[DTNB] (Sigma, D-8130). Total glutathione was measured by firstly reducing oxidized glutathione using glutathione reductase (Sigma, G-3664) and β-NADPH (Sigma, N-7505) followed by conjugation with DTNB. To measure GSSG, GSH was firstly covalently reacted with 2-vinylpyridine (Aldrich, 132292), then GSSG was measured as described above. Pierce® BCA protein assay kit was used for protein determination. All samples were run in triplicates. GSH and GSSG levels were expressed in nmol/mg protein.
G3664
Manufactured by Merck Group
Sourced in Germany
G3664 is a laboratory equipment product offered by Merck Group. It serves as a core function in various laboratory settings, providing a reliable and efficient solution for specific applications. The detailed description of its intended use and functionality is not available in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
G4251, by Merck Group (2 mentions)
D8130, by Merck Group (1 mentions)
N7505, by Merck Group (1 mentions)
β nadph, by Merck Group (1 mentions)
L glutathione reduced, by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Nadph, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Carl Roth (1 mentions)
7 protocols using g3664
1
Glutathione Measurements in Cell Cultures
2
Glutathione Peroxidase Activity Assay
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Glutathione peroxidase (GPX) activity was measured based on the oxidation of reduced glutathione (GSH) by GPX coupled to the disappearance of NADPH catalyzed by glutathione reductase (GR).31 , 34 The rate of NADPH oxidation was monitored spectrophotometrically at 340 nm. Briefly, 2 assays (A & B) were prepared each containing 0.1 M K2HPO4/1 mmol/L EDTA (pH 7.0), 10 mmol/L L‐glutathione reduced (G4251, Sigma, MO), 2.4 unit/mL glutathione reductase (G3664, Sigma, MO). The non‐enzymatic and H2O2‐independent NADPH depletion were subtracted from the total GPX activity, by comparing the absorbance changes after addition of H2O2 in the assays. Activities were normalized to the added lysate volume and protein concentration.
3
Spectrophotometric Determination of GPx1
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Lawrence and Burk’s method was used to determine glutathione peroxidase 1 (GPx1) spectrophotometrically [51 (link)]. Samples and standards were measured in a buffer containing reduced glutathione (Sigma-Aldrich, G4251, Saint Louis, MO, USA) (1 mM), glutathione reductase (Sigma-Aldrich, G3664, Saint Louis, MO, USA) (1 EU/mL), and NADPH (Sigma-Aldrich, 10107824001) (0.2 mM). The activity was determined by measuring the disappearance of NADPH at 340 nm following the addition of H2O2 (final concentration = 0.25 mM). Furthermore, 1 μmole of NAPDH oxidized/min was used to define 1 unit of GPx1. The activity was then normalized to protein content in the sample as measured using the DC Protein Assay Kit.
4
Glutathione Peroxidase Activity Assay
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A measurement of GPx activity was performed according to Flohé and Günzler (1984) (link) with some modifications (Fontaine et al., 1994 ) as described by Krasuska and Gniazdowska (2012) (link). An enzymatic extract (25 µl) was incubated with a 0.2-ml reaction mixture: 0.05-M potassium phosphate buffer (pH 7.0) with 0.1-M aminotriazole, 2.5-mM EDTA, 1.25-mM GSH, and 1.5 U of GR (Sigma-Aldrich, G3664) at 25°C for 10 min. After incubation, 50 µl of 2-mM H2O2 was added. The reaction was started by adding 25 µl of 2.5-mM NADPH. The GPx activity was determined as an absorbance decrease monitored at 340 nm using a microplate reader (Sunrise, Tecan). The activity was expressed as nanomoles NADPH per minute per microgram protein.
5
Glutathione Assay Protocol
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Total glutathione (expressed as GSH equivalents: 2GSH + GSSG) levels were assessed in cellular lysates by the Tietze recycling assay [20 (link)]. Oxidized glutathione (GSSG) was measured by reacting samples with 2-vinylpyridine for 1 h. The reaction was then terminated with triethanolamine (TEA). 3 to 10 μg of tissue homogenate or cell lysate was loaded into a 96-well plate; no more than 20 μg was loaded for GSSG. Absorbance was monitored at 412 nm over 2 minutes in the presence of 0.5 mM dithionitrobenzene (DNTB), 0.24 mM NADPH, and GSH reductase (1 : 3000) in ammonium sulfate (G3664, Sigma). Standard curves were produced for both GSH and GSSG. Linear regression analysis was used to calculate GSH concentrations that were then normalized to the protein concentration.
6
Quantifying Grx Activity via HED Assay
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The hydroxyethyl disulfide (HED) assay34 (link) was performed in a 96-well plate format. The final reaction mixtures of 200 µl contained 100 mM potassium phosphate buffer (pH 7.8), 200 µM NADPH, 1 mM GSH, 3 × 10−3 g l−1 glutathione reductase from yeast (G3664, Merck, Darmstadt, Germany), and variable concentrations of HED (0–1 mM). The concentration of the Grxs was optimized for all proteins individually and lay between 0.5 and 62.5 µg ml−1. The assay was run for 20 min. The linear range of the decrease in absorption was determined for each reaction individually.
7
Glutathione Redox Assay Protocol
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