Gsh glo reagent

Manufactured by Promega

Sourced in Japan

The GSH-Glo Reagent is a luminescent-based assay designed to quantify reduced glutathione (GSH) levels in cells and tissues. The reagent utilizes a GSH-specific probe that reacts with GSH to produce a luminescent signal proportional to the GSH concentration in the sample.

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Lab products found in correlation

Ros glo h2o2 substrate, by Promega (2 mentions) Glomax 96 microplate luminometer, by Promega (1 mentions) Cellrox orange reagent, by Thermo Fisher Scientific (1 mentions) Enspire 2300 multilabel plate reader, by PerkinElmer (1 mentions) Gsh glo glutathione assay kit, by Promega (1 mentions) White 96 well plate, by Thermo Fisher Scientific (1 mentions) N acetyl l cysteine nac, by Fujifilm (1 mentions)

8 protocols using gsh glo reagent

Quantifying Cellular GSH and ROS Levels

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Regarding cellular GSH measurements, 1 × 10⁴ T24 or T24PR cells in 100 μL of culture medium were plated on each well of a 96-multiwell white plate, allowed to attach for 24 h, and each well was then washed three times with PBS. Following the addition of 100 μL of GSH-Glo Reagent (Promega Corp., Madison, WI) at room temperature for 30 min, 100 μL of the luciferin detection reagent was added at room temperature for an additional 15 min. The luminescence intensity of each well was recorded on a GloMax™ 96 Microplate Luminometer (Promega Corp., Madison, WI).
In cellular ROS measurements, 1 × 10⁴ T24 or T24PR cells in 100 μL of culture medium were plated on each well of a 96-multiwell white plate for 24 h and were treated with various concentrations of CDDP for 24 h. Cellular H₂O₂ was assessed by adding 20 μL of the ROS-Glo H₂O₂ substrate (Promega Corp., Madison, WI) to each well, which were then left standing at 37 °C for 2 h in a humidified 5% CO₂ atmosphere. A 100 μL aliquot of ROS-Glo detection solution was added to the resulting mixture and incubated at room temperature for 20 min. The luminescence intensity of each well was recorded on a GloMax™ 96 Microplate Luminometer.

Hagiwara M., Kikuchi E., Tanaka N., Kosaka T., Mikami S., Saya H, & Oya M. (2018). Variant isoforms of CD44 involves acquisition of chemoresistance to cisplatin and has potential as a novel indicator for identifying a cisplatin-resistant population in urothelial cancer. BMC Cancer, 18, 113.

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Quantification of Cellular Redox Status

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For cellular GSH measurements, 1 × 10⁴ cells in 100 μl of culture medium were plated on a 96‐well white plate and allowed to attach for 24 h. Following the addition of 100 μl of GSH‐Glo Reagent (Promega Corp, Tokyo, Japan) at room temperature for 30 min, 100 μl of the luciferin detection reagent was added at room temperature for an additional 15 min. The luminescence intensity of each well was recorded on a GloMax™ 96 Microplate Luminometer.
Intracellular ROS levels were assessed using CellROX orange reagent (C10443; Invitrogen, USA). Cells were seeded at a density of 1 × 10⁴ cells in 100 μl of culture medium per well in a 96‐well black plates to reach 80% confluence. For cellular ROS measurements, T24 (WT, GR, GR #siNTC, GR #siIDH2#1, GR #siIDH2#2, and GR with AGI6780) and UMUC3 (WT, GR, GR #siNTC, GR #siIDH2#1, GR #siIDH2#2, and GR with AGI6780) cells were treated with control vehicle, CDDP 1 μM, and CDDP 10 μM for 3 h and stained with 5 μM CellROX orange reagent at 37°C for 30 min. The cells were washed twice with PBS and replaced with fresh medium for living image of intracellular ROS measurement. ROS levels were quantified as a measure of fluorescence at 545/565 nm by using 2300 EnSpire Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA).

Shigeta K., Hasegawa M., Hishiki T., Naito Y., Baba Y., Mikami S., Matsumoto K., Mizuno R., Miyajima A., Kikuchi E., Saya H., Kosaka T, & Oya M. (2023). IDH2 stabilizes HIF‐1α‐induced metabolic reprogramming and promotes chemoresistance in urothelial cancer. The EMBO Journal, 42(4), e110620.

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Intracellular GSH and ROS Quantification

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Intracellular GSH levels were examined using a GSH‐Glo Glutathione Assay kit (Promega). MBT‐2V cells treated with SSZ (300 or 400 μmol/L), BSO (100 μmol/L), or the vehicle control for 24 hours were harvested and diluted in PBS to 5 × 10³ cells/well in 96‐well white plates. GSH‐Glo Reagent (Promega) was added to the plates and incubated for 30 minutes. Reconstituted Luciferin Detection Reagent (Promega) was added to the wells and incubated for 15 minutes. The luminescence signal was detected using an Enspire 2300. The experiment was carried out in triplicate, and data were expressed as the mean ± SE of intracellular GSH concentrations.
Intracellular ROS levels were assessed using dichlorodihydrofluorescein (DCF) fluorescence staining (C6827; Invitrogen, Tokyo, Japan). MBT‐2V cells treated with SSZ (300 or 400 μmol/L), BSO (100 μmol/L), or the vehicle control were harvested and diluted in PBS to 1 × 10⁵ cells/well in 6‐well plates for 24 hours. These cells were incubated with 10 μmol/L dichloro‐dihydro‐fluorescein diacetate at 37°C for 15 minutes and washed twice with PBS. The mean intensity of DCF fluorescence in 1 × 10⁴ cells was assessed using flow cytometry. The results obtained were analyzed using Image StreamX/Flow Sight (Merck Millipore, Burlington, MA, USA). The experiment was carried out in triplicate, and data were expressed as the mean ± SE of intracellular ROS levels.

Ogihara K., Kikuchi E., Okazaki S., Hagiwara M., Takeda T., Matsumoto K., Kosaka T., Mikami S., Saya H, & Oya M. (2019). Sulfasalazine could modulate the CD44v9‐xCT system and enhance cisplatin‐induced cytotoxic effects in metastatic bladder cancer. Cancer Science, 110(4), 1431-1441.

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Nanoparticle-Induced Oxidative Stress Assay

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There were 1 × 10⁴ BEAS-2B or RAW 264.7 cells in
100 μL of culture medium plated in each well of a 96 multiwell
white plate (Costar, Corning, NY) overnight. The medium was removed,
and cells were treated with 100 μL of 50, 100, and 200 μg/mL
of each nanoparticle suspension for the indicated time period. Cellular
H₂O₂ was assessed 4 h after addition of the
particles by adding 20 μL of ROS-Glo H₂O₂ substrate (Promega Corp.) to each well and then left standing for
2 h at 37 °C in a humidified 5% CO₂ atmosphere. A
100 μL amount of ROS-Glo detection solution was added to the
resulting mixture and incubated for 20 min at room temperature. The
luminescence intensity of each well was recorded on a SpectraMax M5
microplate reader. For cellular GSH assessment, the culture medium
was removed following 6 h of incubation with the particles, and each
well was washed three times with PBS. Following addition of 100 μL
of GSH-Glo reagent (Promega Corp.) for 0.5 h at room temperature,
100 μL of Luciferin Detection Reagent was added for an additional
15 min at room temperature. The luminescence intensity of each well
was recorded on a SpectraMax M5 microplate reader.

Zhang H., Pokhrel S., Ji Z., Meng H., Wang X., Lin S., Chang C.H., Li L., Li R., Sun B., Wang M., Liao Y.P., Liu R., Xia T., Mädler L, & Nel A.E. (2014). PdO Doping Tunes Band-Gap Energy Levels as Well as Oxidative Stress Responses to a Co3O4p-Type Semiconductor in Cells and the Lung. Journal of the American Chemical Society, 136(17), 6406-6420.

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Cellular Glutathione and Reactive Oxygen Species Measurement

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Regarding cellular GSH measurements, 1 × 10⁴ cells in 100 μL of culture medium were plated on a 96‐multiwell white plate and allowed to attach for 24 h. Following the addition of 100 μL of GSH‐Glo Reagent (Promega Corp, Tokyo, Japan) at room temperature for 30 min, 100 μL of the luciferin detection reagent was added at room temperature for an additional 15 min. The luminescence intensity of each well was recorded on a GloMax™ 96 Microplate Luminometer. Intracellular ROS levels were assessed using dichlorodihydrofluorescein (DCF) fluorescence staining (C6827; Invitrogen). Regarding cellular ROS measurements, T24WT, T24CR, UMUC3WT, and UMUC3CR cells were treated with control vehicle and CDDP at 10 μmol/L for 24 h, and then harvested and diluted in PBS to 1 × 10⁵cells/well in 6‐well plates for 24 h. These cells were incubated with 10 μmol/L dichlorodihydrofluorescein diacetate (H₂DCFDA) at 37°C for 15 min and washed twice with PBS. The mean intensity of DCF fluorescence was assessed using Image StreamX/Flow Sign flow cytometry.

Shigeta K., Hasegawa M., Kikuchi E., Yasumizu Y., Kosaka T., Mizuno R., Mikami S., Miyajima A., Kufe D, & Oya M. (2020). Role of the MUC1‐C oncoprotein in the acquisition of cisplatin resistance by urothelial carcinoma. Cancer Science, 111(10), 3639-3652.

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Glutathione Assay for Dose-Dependent DC10 Effects

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5000 cells from the A172, A375 and MCF7 cell lines were suspended in 100 µL that was loaded in each well of a white 96-well plate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and allowed to incubate for 24h. The medium was removed and replaced with an increasing concentration of DC10 from 25-1000 µM After 48 h of incubation, the media was removed and GSH-Glo™ reagent (Promega, Cat#: V6911) was added followed by 30 min incubation. 100 μL reconstituted luciferin detection reagent was added and incubated for another 15 min. The bioluminescence was quantified with the Victor3™ plate reader (PerkinElmer, Waltham, Massachusetts, USA) as counts per second with an integration time of one second. The averages of triplicate readings were exported to the GraphPad Prism software and expressed as fold change to DMSO controls. This procedure was repeated in separate experiments measuring GSH levels in the same cell lines without and with 10 mM NAC, untreated or treated with 50 μM 100 μM and 150 μM doses of DC10. All experiments were conducted three times.

Sarowar S., Cirillo D., Játiva P., Nilsen M.H., Otragane S.M., Heggdal J., Selheim F., Ceña V., Bjørsvik H.R, & Enger P.Ø. (2022). The Styryl Benzoic Acid Derivative DC10 Potentiates Radiotherapy by Targeting the xCT-Glutathione Axis. Frontiers in Oncology, 12, 786739.

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Quantification of Cellular GSH and ROS

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Cells were seeded in dark-colored, flat-bottomed 96-well plates. In GSH assays, cells at a density of 5 x 10³ cells/well were treated with GSH-Glo reagent (Promega, Madison, WI, USA) as stipulated by the manufacturer and luminescence was analyzed in a microplate reader. In ROS assays, cells at a density of 2 x 10⁴ cells/well were stained with 10 μM of dichlorodihydrofluorescein diacetate (DCF-DA) (Sigma-Aldrich, St. Louis, MO, USA) for 45 minutes, treated with a ROS inducer (5 μM of CDDP) and/or ROS scavenger [5 or 10 mM of N-Acetyl-L-cysteine (NAC) (Wako, Osaka, Japan)] for 4 hours, and then analyzed in a microplate reader. We utilized a previously described protocol for GSH and ROS analyses [18] (link). Data was representative of three independent experiments with 4 replicates.

Mvunta D.H., Miyamoto T., Asaka R., Yamada Y., Ando H., Higuchi S., Ida K., Kashima H, & Shiozawa T. (2017). SIRT1 Regulates the Chemoresistance and Invasiveness of Ovarian Carcinoma Cells. Translational Oncology, 10(4), 621-631.

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Glutathione Quantification Assay

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Cells were seeded in 96-well plates overnight; 100 μL of the GSH-Glo reagent (Promega, Madison, WI) was added to each well, mixed, and the plate was incubated at room temperature for 30 minutes. Luciferin Detection Reagent was added to each well and luminescence was recorded.

Das S., Jackson W.P., Prasain J.K., Hanna A., Bailey S.K., Tucker J.A., Bae S., Wilson L.S., Samant R.S., Barnes S, & Shevde L.A. (2017). Loss of Merlin induces metabolomic adaptation that engages dependence on Hedgehog signaling. Scientific Reports, 7, 40773.

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