MMCs were seeded in 96-well plates and were treated with deltamethrin (0–5 µM) ± salubrinal (50 µM) for 24 and 48 h. Following the treatment, cells were harvested in lysis buffer and then the concentration of glutathione was measured using a glutathione assay kit (Cat #AB65322, Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions. Briefly, samples and GSH standards were incubated with glutathione S-transferase (GHT) and monochlorobimane (MCB) for 60 min at 37 °C and then fluorescence was measured with excitation 380 nm and emission 461 nm using a SpectraMax M5 (Molecular Devices, LLC., San Jose, CA, USA) microplate reader. The concentration of GSH in the samples was calculated as nmol/mg protein. Protein content was determined using PierceTM BCA protein assay kit (Cat #23225, Thermo Fisher Scientific, Rockford, IL, USA).
Glutathione assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Glutathione Assay Kit is a colorimetric assay designed to quantify the total glutathione levels, both reduced (GSH) and oxidized (GSSG), in a variety of sample types. The kit provides a simple, rapid, and sensitive method to measure glutathione concentration.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Mda assay kit, by Abcam (2 mentions)
Iron assay kit, by Abcam (2 mentions)
α tubulin, by Merck Group (2 mentions)
Nb300 318, by Novus Biologicals (2 mentions)
Phospho eif2α, by Abcam (2 mentions)
Total eif2α, by Merck Group (2 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Fluorescence microplate reader, by Thermo Fisher Scientific (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Sod assay kit, by Abcam (1 mentions)
Ab118970, by Abcam (1 mentions)
Cell lysis buffer, by Solarbio (1 mentions)
Cell counting kit 8 (cck8), by AbMole (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Atp bioluminescence assay kit cls 2, by Roche (1 mentions)
Fluorometric assay kit, by Abcam (1 mentions)
Oxiselect glutathione assay kit, by Cell Biolabs (1 mentions)
Glutathione detection kit, by Abcam (1 mentions)
Imark microplate absorbance reader, by Bio-Rad (1 mentions)
25 protocols using glutathione assay kit
1
Glutathione Quantification in Deltamethrin-Treated Cells
2
Glutathione Assay Protocol
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The assay was performed according to the manufacturer′s instructions (Abcam Plc., Cambridge, UK) using a Glutathione Assay Kit (Abcam Plc.). The results were expressed relative to 100% of the control group.
3
Glutathione Assay for Oxidative Stress
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Cells were plated in 100 mm dishes and treated with DOX and 8-OHdG for 24 h. A glutathione assay kit (Abcam, Cambridge, UK) was used as per the manufacturer’s instructions. Briefly, cell suspension from lysed 106 cells using lysis buffer was incubated in medium containing thiol or GSSG probe at room temperature for 10–60 m. Then, we measured concentrations of GSH and total glutathione (GSH + GSSG) at Ex/Em = 490/520 nm using a fluorescence microplate reader (Thermo Scientific, Waltham, MA, USA). The GSH/GSSG ratio was calculated from concentrations of GSH and GSSG.
4
Evaluating Oxidative Stress Markers
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SOD activity, glutathione peroxidase (GPx) activity, lipid peroxidation of MDA, and LPO in plasma were measured by using SOD Assay Kit (ab65354), Glutathione Assay Kit (ab102530), MDA Assay Kit (ab118970), and LPO Assay Kit (ab133085), respectively, according to the manufacturer’s instructions (Abcam, Cambridge, MA, USA). The optical density for SOD, GPx, MDA, and LPO kits was read at 450, 340, 532, and 500 nm wavelengths, respectively, using an Epoch microplate spectrophotometer (BioTek® Instruments, Inc. Highland Park, VT, USA). The intra-assay CV for SOD, GPx, MDA, and LPO was 2.9%, 0.1%, 1.3%, and 1.1%, respectively. The inter-assay CV for SOD and GPx was 2.7% and 0.1%, respectively.
5
VEGF and GSH Quantification in Cells
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The method of processing cells and tumor tissues was as described above. The VEGF protein levels were measured according to the manufacturer’s instructions by using an enzyme-linked immunosorbent assay kit (ELISA, R&D Systems, Minneapolis, MN, USA). GSH was determined by using Glutathione Assay Kit (Abcam, ab65322). Final VEGF and GSH levels were normalized to the total protein concentration.
6
Antioxidant and Oxidative Stress Assays
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The levels of GSH in cell extracts were analyzed using the Glutathione Assay kit (Abcam, cat. no. ab239727). Total glutathione/oxidized glutathione (GSH/GSSG) was determined using the Total GSH/GSSG Assay kit (Abcam, cat. no. ab138881). MDA levels were determined using a Lipid Peroxidation (MDA) Assay kit (Abcam, cat. no. ab118970). The activity of SOD was measured with the Superoxide and Dismutase Assay kit (Solarbio, cat no. BC0170). The content of iron in cells or tissue lysates was detected using the Iron Assay Kit (Abcam, cat. no. ab83366).
7
Glutathione Quantification in Murine Liver
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The GSH level in mice liver tissue was measured using a Glutathione Assay Kit (Abcam, ab235670). All procedures were according to the manufacturer’s protocols.
8
Glutathione Quantification in NASH Mouse Liver
9
Lipid Peroxidation and GSH Assay
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MDA in BMDMs and RAW264.7 cells was analyzed using a lipid peroxidation assay kit (ab118970; Abcam, Cambridge, England) in the presence or absence of ZA (50 μM). GSH content in BMDMs and RAW264.7 cells was assayed using a Glutathione Assay Kit (ab65322; Abcam, Cambridge, England) according to the standard protocol. Briefly, cell supernatant, 5, 5′-dithio-bis 2-nitrobenzoic acid solution and the reagents of kits were mixed together and incubated at RT for 10 min, then NADPH was added into this system to trigger the reaction. The absorbance of 5-thio-2-nitrobenzoic acid was detected at 412 nm.
10
Decursin Effects on HSC Viability and Oxidative Stress
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LX-2 cell viability was measured with Cell Counting Kit-8 (AbMole, USA). Cells (4 × 10 3 cells/well) were seeded into 96-well plates to incubate for 1 day, and then cells were treated with decursin (30 μmol/L) for 72 h. About 10 μL of CCK-8 reagent was applied to treat cells for 60 min. The absorbance was calculated using Automatic Enzyme Marker (Tecan, Männedorf, Switzerland) at 450 nm.
Fluorescein diacetate staining TGF-β (5 ng/mL)-activated LX-2 cells were treated with decursin (30 μmol/L). After washing with phosphate-buffered saline (PBS), LX-2 cells were incubated with fluorescein diacetate (FDA) solution (10 mg/mL, Sigma-Aldrich) for 0.5 h at room temperature. Then cells were photographed with a fluorescence microscope (Olympus Corporation, Japan).
Glutathione and Fe 2+ assay LX-2 cells were activated with TGF-β (5 ng/mL) in the presence or absence of decursin (30 μmol/L). Around 72 h later, cells were homogenized in cell lysis buffer (Solarbio), and then supernatant was collected from cell lysates by centrifuging for 10 min at 4 • C at top speed. The levels of glutathione (GSH) and Fe 2+ were assessed with a Glutathione Assay Kit (ab65322, Abcam) and iron assay kit (ab83366, Abcam), respectively, according to the manufacturers' instruction.
Fluorescein diacetate staining TGF-β (5 ng/mL)-activated LX-2 cells were treated with decursin (30 μmol/L). After washing with phosphate-buffered saline (PBS), LX-2 cells were incubated with fluorescein diacetate (FDA) solution (10 mg/mL, Sigma-Aldrich) for 0.5 h at room temperature. Then cells were photographed with a fluorescence microscope (Olympus Corporation, Japan).
Glutathione and Fe 2+ assay LX-2 cells were activated with TGF-β (5 ng/mL) in the presence or absence of decursin (30 μmol/L). Around 72 h later, cells were homogenized in cell lysis buffer (Solarbio), and then supernatant was collected from cell lysates by centrifuging for 10 min at 4 • C at top speed. The levels of glutathione (GSH) and Fe 2+ were assessed with a Glutathione Assay Kit (ab65322, Abcam) and iron assay kit (ab83366, Abcam), respectively, according to the manufacturers' instruction.
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