Intracellular reduced glutathione levels were determined using commercially available fluorometric thiol probe thiol tracker Violet (Thermo Fisher) and the total glutathione (GSH) Detection Assay Kit (Abcam, MA, USA). CD4+ T cells were stained with thiol‐tracker violet (10 µm ) for 30 min at 37 °C. Cells were washed twice with PBS, stained with surface antibodies of interest (i.e., α‐CD3, α‐CD4, and α‐CD69; 25 µg mL−1) for 20 min, fixed in 2% paraformaldehyde, and data collected by flow cytometry. The total GSH detection assay was performed according to the manufacturer's directions.
Gsh detection assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The GSH (Glutathione) Detection Assay Kit is a colorimetric assay designed to measure the total glutathione (GSH) levels in various biological samples. The kit provides a simple, rapid, and sensitive method for the quantitative determination of GSH.
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Lab products found in correlation
Thioltracker violet, by Thermo Fisher Scientific (1 mentions)
α ketobutyrate, by Merck Group (1 mentions)
Tempol, by Bio-Techne (1 mentions)
Lactate glo assay, by Promega (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Pyruvate assay kit, by Abcam (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Glomax, by Promega (1 mentions)
Ros assay kit, by Cell Biolabs (1 mentions)
Facs lsrfortessa, by BD (1 mentions)
Monochlorobimane, by Merck Group (1 mentions)
L buthionine sulfoximine bso, by Merck Group (1 mentions)
Antibiotic strips, by bioMérieux (1 mentions)
Palcam agar, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
9 protocols using gsh detection assay kit
1
Intracellular Glutathione Quantification
2
Metabolic Profiling of Thymocytes in TBI
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Thymuses from untreated and SL-TBI treated mice were harvested and enzymatically digested and stained from flow cytometry analysis of thymocyte populations as above. Further analysis of mitochondrial bioenergetics were assessed using TMRE (Abcam, ab113852), MitoTracker™ Green FM (Invitrogen, M7514), MitoSOX™ Red Mitochondrial Superoxide Indicator (ThermoFisher, M36008), and Intracellular glutathione (GSH) Detection Assay Kit (Abcam, ab112132). Thymocytes were isolated from untreated and TBI-treated mice and intracellular pyruvate and lactate levels were measured by absorbance using Pyruvate Assay kit (Abcam, ab65342) or Lactate-Glo™ Assay (Promega, J5021). Thymocytes were isolated from untreated and TBI-treated mice and incubated in RPMI with 5 mM sodium pyruvate (Gibco, 11360070) for 3 h at 37 °C and stained for flow cytometry analysis and cl-caspase 1 levels. Cells were further incubated with 5 mM sodium pyruvate plus 200 μM α-ketobutyrate (Sigma-Aldrich) or 100 μM TEMPOL (Tocris, 3082) for 3 h and cells were prepared for flow cytometry analysis.
3
Glutathione Quantification Assay Protocol
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GSH concentration was calculated using the GSH Detection Assay Kit (Abcam; ab138881) as detailed in the manufacturer’s protocol. Briefly, 2.5×105 cells (p53 or N1IC of similar passage) were plated per well of a six-well plate and grown for 24 hours as detailed above. Cells were washed with cold PBS and lysed with 100 μL ice-cold 1× Mammalian Lysis Buffer. Lysates were centrifuged at 21,000 g for 15 min. The supernatant was collected, mixed with 20 μL ice-cold trichloroacetic acid, incubated on ice for 10 minutes and centrifuged at 12,000 g for 5 minutes. The supernatant was neutralized by addition of NaHCO3 to a final pH of 6, centrifuged at 13,000 g for 5 minutes, and supernatants collected. Samples were corrected for the diluted volumes, mixed with GSH Assay Mixture and GSH was calculated by measuring in a microplate reader (Promega GloMax) using 490/520 nm excitation/emission filter and fitting into a GSH standard curve.
4
GSH Quantification in MBMEC Cells
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MBMEC were cultured in 24-well plates. GSH levels were measured using a GSH detection assay kit from Abcam, according to the manufacturer´s instructions.
5
Oxidative Stress and Inflammatory Markers Evaluation
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Reduced Glutathione (GSH) concentration and reactive oxygen species (ROS) concentration were measured using the GSH detection assay kit (Abcam Inc., Cambridge, MA, U.S.A.) and the ROS assay kit (Cell Biolabs, Inc., San Diego, CA, U.S.A.), respectively. SOD activity was measured spectrophotometrically with the Dojindo SOD Activity Kit-WST (Dojindo Molecular Technologies, Rockville, MD, U.S.A.). Malondialdehyde (MDA) concentration in plasma was determined in terms of thiobarbituric acid reactive substances (TBARS) formation as described by Ohkawa et al. [27 (link)]. Plasmatic C-reactive protein (CRP), TNF-α, IL-1β, IL-6, and IL-10 concentrations were assayed using commercial ELISA kits (R & D systems Inc., Minneapolis, MN, U.S.A.) following manufacturer.
6
Intracellular GSH Quantification by Flow Cytometry
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Intracellular level of GSH was measured by intracellular GSH Detection Assay Kit (Abcam). About 106 control, overexpressed, KO, and complement cells were taken and washed the cells with twice with 1× PBS at 1500g for 6 min. Five microliters of 200× Thiol Green Dye was mixed into 1 ml of cell suspension and incubated at 22 °C for 15 min, then washed the cells at 1000 rpm for 4 min and resuspended in 1 ml of assay buffer. Fluorescence intensity was measured by flow cytometer (BD FACS LSR Fortessa) (Exλ-490 nm; Emλ-525 nm).
7
Monitoring Reduced Glutathione in HK-2 Cells
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Reduced GSH in HK-2 cells treated with BiZn (10 μM and 100 μM) for 24 and 48 hours was monitored using monochlorobimane assay (Sigma, USA). This probe reacts specifically with reduced GSH through GSH transferase to form a fluorescent derivative [28] (link). To investigate the GSH production, HK-2 cells were resuspended in DMEM with 10% FCS. BiZn (10 μM or 100 μM) and L-buthionine-sulfoximine (BSO) (250 μM or 500 μM) (Sigma, USA) were added to the cells respectively for 24 or 48 hours. The cells were treated with monochlorobimane (Sigma, USA) at a final concentration of 100 mM in Ca2+- and Mg2+-free PBS for 0 to 60 minutes at 37°C. The fluorescence produced by the cells was measured with a fluorometer at an excitation wavelength of 360 nm and emission wavelength of 480 nm.
Reduced GSH in samples was measured by using GSH Detection Assay Kit (Abcam). Briefly, HK-2 cells were treated with BiZn (10 μM and 100 μM) for 48 hours. A total of 0.2 × 106 cells were homogenized by Mammalian Lysis Buffer. Enzymes were removed by using Deproteinizing Sample Kit (Abcam). Nonfluorescent GSH Assay Mixture reacts with reduced GSH in cell lysate to become fluorescent. The fluorescence produced was measured with a fluorometer at an excitation wavelength of 490 nm and emission wavelength of 520 nm.
Reduced GSH in samples was measured by using GSH Detection Assay Kit (Abcam). Briefly, HK-2 cells were treated with BiZn (10 μM and 100 μM) for 48 hours. A total of 0.2 × 106 cells were homogenized by Mammalian Lysis Buffer. Enzymes were removed by using Deproteinizing Sample Kit (Abcam). Nonfluorescent GSH Assay Mixture reacts with reduced GSH in cell lysate to become fluorescent. The fluorescence produced was measured with a fluorometer at an excitation wavelength of 490 nm and emission wavelength of 520 nm.
8
Detailed Reagent and Cell Line Sources
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Antibiotic strips were purchased from BioMérieux (Marcy-l’Étoile, Lyon, France). Palcam agar was purchased from Oxoid (CM0877; Hampshire, UK). Sheep blood was obtained from MBCell (Seoul, Korea). De Man, Rogosa, and Sharpe (MRS) agar and broth were purchased from Difco (Detroit, MN, USA). HepG2 and HT-29 cells were obtained from the Korea Cell Line Bank (Seoul, Korea). Minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin–streptomycin (PS) were purchased from Gibco (Grand Island, NY, USA). Thiobarbituric-acid-reactive substance (TBARS) and GSH assay kits were purchased from Cayman (Ann Arbor, MI, USA). Mouse malondialdehyde (MDA) ELISA kit was obtained from LSBio (Seattle, DC, USA). GSH detection assay kit and CAT, SOD, and GPx assay kits were purchased from Abcam (Cambridge, UK). NuceloZOL for mRNA extraction was obtained from Macherey-Nagel GmbH & Co. (Dueren, Germany). The reverse transcriptase premix was purchased from Intron (Seongnam-si, Korea). The iQ™ SYBR® Green Supermix was purchased from Bio-Rad (Hercules, CA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Flow Cytometric Analysis of Intracellular ROS and GSH
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Flow cytometry was used for the detection of the intracellular ROS and GSH levels using the DCF-DA and intracellular glutathione (GSH) Detection Assay Kit (ab112132; Abcam, Cambridge, UK), respectively. Furthermore, 200,000 cells/well were seeded in two separate 6-well plates, one for ROS and one for GSH detection. Cells were treated in the presence of increasing concentrations of the extract for 24 h in serum-free medium. After the treatment, ROS detection was performed by placing 1 mL of 10 μΜ DCF-DA (20 mM stock in DMSO) in PBS in each well after incubation for 45 min at 37 °C. After this step, the cells were trypsinized, centrifuged (1200 rpm, 5 min, 4 °C), and re-suspended in 250 μL of PBS. Simultaneously, GSH detection was performed according to the manufacturer’s instructions. Briefly, cells were trypsinized and re-suspended in 1 mL of PBS with 5 μL of green dye and incubated for 30 min at 37 °C in the dark. Next, cells were centrifuged (1200 rpm, 5 min, 4 °C) and re-suspended in 250 μL of PBS. Then, all samples were submitted to flow cytometric analysis using a FACScan flow cytometer (Becton Dickinson, NJ, USA) with excitation and emission length at 490/520 nm for ROS and for GSH detection.
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