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Gsh glo glutathione assay

Manufactured by Promega
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The GSH-Glo Glutathione Assay is a luminescent-based assay that quantifies the amount of glutathione in biological samples. It utilizes a luminogenic substrate that reacts with glutathione to generate a luminescent signal, which is proportional to the amount of glutathione present in the sample.

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101 protocols using gsh glo glutathione assay

1

Glutathione Concentration Quantification

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The relative GSH concentration in cell lysates was detected using a GSH-Glo Glutathione Assay (V6911; Promega) according to the manufacturer’s instructions. Briefly, 5,000 cells/well were seeded in a 96-well plate 1 d before adding drugs. The culture medium from the wells was carefully removed and 100 μl of GSH-Glo Reagent was added and incubated at room temperature for 30 min on a plate shaker. Finally, 100 μl of reconstituted Luciferin Detection Reagent was added to each well, mixed well, and incubated for 15 min. The luminescence was measured by a SYNEGY|H1 microplate reader (BIOTEK).
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2

Hydrogen Peroxide and Glutathione Assays

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The ROS-Glo™ Assay (Promega Inc. Madison, WI, USA) was used to measure the level of hydrogen peroxide (H2O2) directly in cell culture according to the manufacturer’s recommendations. Glutathione (GSH) depletion was evaluated using the GSH Glo™ Glutathione Assay (Promega, Madison, WI, USA) according to manufacturer’s protocol.
BiP, CHOP or caspase 3 expression was assessed by western blotting as described above for the FATPs (section 2.4). To detect BIP or caspase 3, 30 µg protein from a cleared lysate was separated by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane and then incubated simultaneously with primary antibodies against β-actin (Sigma-Aldrich, St. Louis, MO, USA) and the antibody of interest. In the case of CHOP, 60 µg lysate protein was used per sample. The rabbit polyclonal antibody against caspase 3, BIP or the mouse polyclonal antibody against CHOP protein were purchased from Cell Signaling Technology (Beverly, MA, USA). Protein quantification was performed with the Odyssey system software.
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3

Quantifying Cellular Glutathione Levels

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Glutathione levels were measured using GSH-Glo Glutathione Assay (Promega) according to the manufacturer's instructions. Cells were diluted to 100,000 cells/ml. 50 μl cell suspension was placed in an opaque-walled 96-well plate in triplicate. 50 μl of freshly prepared GSH-Glo Reagent 2X Mix (10 μl luciferin-NT substrate, 10 μl glutathione-s-transferase with 480 μl GSH-Glo reagent) was added to each well in the dark and placed on a plate shaker for 30 seconds prior to incubation at room temperature for 30 min in the dark. After this, 100 μl/well of reconstituted Luciferin Detection Reagent was added and placed on a plate shaker for 30 seconds prior to incubation for 15 min and reading the luminescence. Triplicate values were averaged and the culture media background was subtracted from the average of each experimental sample.
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4

Assessing Cellular Redox Status

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GSH-Glo Glutathione Assay and NADP/NADPH-Glo Assay (both from Promega) were used to measure the levels of GSH and NADPH/NADP+ (total and ratio), respectively, following the manufacturer’s instructions.
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5

Intracellular Glutathione Quantification

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After treatment, cells were harvested by trypsinization and counted using a Z2 Coulter counter. Intracellular reduced glutathione was determined per 10,000 viable cells using the ultrasensitive luminescent GSH-Glo™ glutathione assay (Promega, Madison, WI). Luminescence reading was performed using a BioTek Synergy 2 Reader (BioTek, Winooski, VT, USA). Data represent relative levels of glutathione normalized for cell number comparing treated versus untreated controls (means ± SD; n=3).
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6

Quantification of Cellular Glutathione Levels

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Total extracellular and intracellular glutathione (GSH + GSSG) concentrations were determined using the GSH-Glo glutathione assay (Promega, Madison, WI) per manufacturer’s instruction. Media was collected for analysis of the extracellular GSH ([GSH]e) and GSH-Glo reaction buffer was added to the cells for analysis of the intracellular GSH ([GSH]i). Additional reaction buffer was made per manufacturer’s instructions and 200 µL was added per well as a dilution factor to keep relative light units within the dynamic range of the standards. To determine total GSH levels, glutathione disulfide (GSSG) within the samples was converted to GSH with the reducing agent TCEP-HCl (final concentration = 1 mM; 10 min; 25°C; Thermo Scientific; Waltham, MA). Luciferase activity was measured using an Optocomp II luminometer (MGM Instruments) or a Synergy2 microplate reader (BioTek, Winooski, VT). Total intracellular or extracellular GSH were normalized to standards prepared in GSH-Glo reaction buffer and MS containing 0.1% fatty acid free BSA, respectively. Extracellular GSSG was calculated based on the equation GSSG = (total GSH – reduced GSH) / 2. [GSH]i were normalized to cellular protein. Standards were linear over the range of 0–5 µM.
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7

Glutathione Assay Protocol

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Reduced glutathione levels were assessed as described previously (Aird et al., 2012 (link)) using the GSH-Glo Glutathione Assay (Promega) according to manufacturer’s instructions.
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8

Glutathione Measurement via Luminescence

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Glutathione is one of the most abundant nonenzymatic antioxidants found in cells. Most of the intracellular glutathione is present in the reduced form (GSH), whereas only a small part is oxidized and found as a dimer in which two sulfhydryl groups are connected by a disulfide bond (GSSG).
Changes in GSH levels were monitored with the luminescence-based GSH-Glo glutathione assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Essentially, the assay couples two chemical reactions. The first reaction is catalyzed by glutathione-S-transferase and converts a GSH probe, luciferin-NT, into luciferin. In the second reaction, catalyzed by firefly luciferase, the amount of luciferin produced is detected as a luminescent signal. The overall intensity of the signal is proportional to the GSH amount present in each sample. At the end of exposure to copper and myricetin, the medium was removed, and 100 μL of the GSH-Glo reagent was added to each well. The plates were incubated for 30 min at RT in the dark. Thereafter, 100 μL of the luciferin detection reagent was added, mixed briefly, and incubated for a further 15 min. Emitted luminescence was quantified by using a Fluoroskan Ascent FL luminometer (Thermo Fisher Scientific, Waltham, MA, USA).
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9

Oxidative Stress Measurements in Tumorspheres

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Cells (5 × 103) were seeded per well in six-well ultra-low attachment culture plates (Corning Life Sciences) in DMEM/F12 50/50 medium (Corning Life Sciences) with 20 ng/ml EGF (Millipore Sigma), 20 ng/ml bFGF (Millipore Sigma) and 1% B27 supplement. Cells were treated with vehicle or DOX for 10–14 days. Tumorspheres were counted under an inverted microscope in triplicate wells.
Measurements of ROS, NADP/NADPH, GSH, GSH/GSSG levels. Assays for measurements of ROS (ROS-Glo H2O2 Assay, G8820; Promega, Madison, WI, USA), NADP/NADPH (NADP/NADPH-Glo Assay, G9081; Promega), GSH (GSH-Glo Glutathione Assay, V6911; Promega) and GSH/GSSG (GSH/GSSG-Glo Assay, V6611; Promega) levels were performed according to the manufacturer’s instructions validated with internal controls. Luminescence intensity was detected using an Infinite 200 PRO (Tecan, Madison, WI, USA).
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10

Glutathione Quantification in Infected Cells

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Cells cultured in 96-well tissue culture plates were infected and the GSH levels were measured using a luminescence-based assay (GSH-Glo™ Glutathione Assay, Promega), according to the manufacturer’s instructions.
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