Total cellular GSH (GSx; c[GSx] = c[GSH] + 2 × c[GSSG]) content was determined as described previously (Tietze, 1969 (link)). Briefly, 105 to 106 cells were lysed in ice-cold 1% 5-sulfosiacylic acid. After 30-min incubation on ice, lysates were centrifuged (10 min, 20,000 × g) and the supernatants were used for GSx determination, while the pellets were analyzed for protein content by the BCA assay. Then, 10 μl supernatants were transferred to a 96-well plate and mixed with 100 μl reaction solution containing 0.64 μl glutathione reductase (Sigma) solution, 400 μM NADPH, 300 μM of the colorimetric dye 5,5′-dithiobis-(2-nitrobenzoic acid), and 2 mM EDTA in 100 mM sodium phosphate buffer (pH 7.5). Subsequently, the absorption at 412 nm was followed for 10 to 15 min and the slopes of the resulting curves were determined. GSx concentrations were calculated using standard curves, normalized for protein content and expressed as nmol GSx/mg protein. Depletion of cellular GSx pools was achieved by co-incubation of the cells with 100 μM DMF (Sigma) and 100 μM BSO (Sigma) for up to 4 hr (Boivin et al., 2011 (link)). GSH repletion was performed by co-incubation of cells with BSO/DMF and 2 mM GSH-OEt (Sigma) (Ghoreschi et al., 2011 (link)).
Gsh oet
Manufactured by Merck Group
Sourced in United States
GSH-OEt is a chemical compound used in research and laboratory settings. It serves as a substrate for various enzymatic reactions and has applications in the study of cellular processes and biochemical pathways. The core function of GSH-OEt is to provide a source of glutathione, a key antioxidant, in experimental systems.
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Lab products found in correlation
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Glutathione reductase, by Merck Group (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Ne per extraction reagent kit, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Z ietd fmk, by R&D Systems (1 mentions)
Z lehd fmk, by R&D Systems (1 mentions)
Facscalibur, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
5 protocols using gsh oet
1
Quantification of Cellular Glutathione
2
Oxidative Stress Response Assay in Parasitic Worms
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Freshly isolated adult worms were stabilized for 1 h at 37 °C in serum- and phenol-red-free RPMI medium (catalog No. 11835030, ThermoFisher, Waltham, MA, USA) under 5% CO2 atmosphere. The groups of 10 worms in 1 mL fresh medium were treated with the indicated doses of cumene hydroperoxide (CHP; catalog No. 513296, Sigma-Aldrich; 1–8 mM for 1 h or 4 mM for 0–1 h), buthionine sulfoximine (BSO; catalog No. B2515, Sigma-Aldrich) plus 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU; catalog No. C0400, Sigma-Aldrich) (200 μM each per condition for 1 h), GSH-reduced ethyl ester (GSH-OEt; catalog No. G1404, Sigma-Aldrich; 2 mM for 1 h), and gliotoxin (catalog No. G9893, Sigma-Aldrich; 10–100 μM for 10 min–1 h) at 37 °C. Experimental worms were harvested and separated into nuclear and cytosolic fractions using the NE-PER Extraction Reagent Kit (catalog No. 78835, ThermoFisher) according to the manufacturer’s instructions. Proteins from each compartment were extracted in PBS (100 mM, pH 7.4) containing a protease inhibitor cocktail (1 tablet/25 mL). Nuclear and cytosolic fractions obtained from worms incubated without chemical treatment were used as controls.
3
Antioxidant Modulation of Tobacco-Induced Osteogenic Inhibition
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To counteract tobacco-induced oxidative stress, three antioxidants were used concomitantly with tobacco treatment during days 0–7 of differentiation: ascorbic acid (AA; Sigma-Aldrich) [10 µM], dl-α-tocopherol acetate (vitamin E; Supelco, Sigma-Aldrich) [10 µM], and glutathione reduced ethyl ester (GSH-OEt; Sigma-Aldrich) [500 μM]. The antioxidants were replenished with every medium change.
To explore the involvement of caspases 4, 8 and 9 in tobacco-related inhibition of osteogenic differentiation, tobacco-treated cultures were simultaneously dosed with caspase 4 inhibitor (4i; Z-LEVD-FMK, PromoCell GmbH, Heidelberg, Germany), caspase-8 inhibitor (8i; Z-IETD-FMK, R&D Systems, Minneapolis, MN, USA) or caspase 9 inhibitor (9i; Z-LEHD-FMK, R&D Systems), all at 3 µM, during days 0–7 of differentiation. Inhibitor-supplemented medium was replaced with every medium change.
To explore the involvement of caspases 4, 8 and 9 in tobacco-related inhibition of osteogenic differentiation, tobacco-treated cultures were simultaneously dosed with caspase 4 inhibitor (4i; Z-LEVD-FMK, PromoCell GmbH, Heidelberg, Germany), caspase-8 inhibitor (8i; Z-IETD-FMK, R&D Systems, Minneapolis, MN, USA) or caspase 9 inhibitor (9i; Z-LEHD-FMK, R&D Systems), all at 3 µM, during days 0–7 of differentiation. Inhibitor-supplemented medium was replaced with every medium change.
4
Enhancing Reducing Environment in Cancer Cells
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In order to recapitulate the reducing environment typical of TME by further increasing the GSH level already present in the cancer cells medium, experiments were performed in which the medium was supplemented with glutathione reduced ethyl ester (GSH-OEt, Sigma Aldrich, Saint Louis, MO, USA) [34 (link)]. Cells were seeded as described in 2.11 and pre-treated with 10 mM GSH-OEt for 90 min. At the end of the pre-incubation time, cells were treated with nanoparticles for 5 h and released in drug-free medium for a further 24 h before being assessed for viability with MTS assay. If foreseen, after the 5 h of incubation with nanoparticles, cells were irradiated in PBS with red light (600–750 nm) emitted by a PDT1200 lamp (Waldmann, Villingen-Schwenningen, Germany), and with a total fluence of 1 J/cm2 at a power density of 20 mW/cm2.
5
Glutathione-Responsive Nanogel Uptake in HeLa Cells
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Hela cells were seeded in 6-well plates, until the confluence state. Subsequently, cells were treated with glutathione reduced ethyl ester (GSH-OET, Sigma Aldrich) (10 mM) for 2 h [26 ]. After washing with PBS, cells were incubated at 37 °C for 3 h with P*(0.5)AA50-AEDP-DOXnanogels at final DOX concentration of 1 μg/mL in complete DMEM medium. GSH-OET untreated cells were used as control. Next, cells were washed twice with PBS, detached by Trypsin-EDTA 1×, collected by centrifugation at 1000 rpm for 5 min order to obtain cell pellet that was re-suspended into 0.5 mL of PBS. For each sample were collected 1 × 104 events investigated by FACS-Calibur (Becton Dickinson) using BD FACS Diva software.
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