To measure oxidative stress levels, relative concentration of total glutathione (GSSG/GSH) were determined using the OxiSelectTM Total Glutathione GSSG/GSH Assay kit (Cell Biolabs, Inc., CA, USA) in BON1 cells. 900,000 cells were seeded in 10 cm plates and 24 h later the cells were treated with TH588 (5 μM) alone and in combination with 10 nM everolimus or 5 μM 5-FU. After 96 h of incubation the cells were collected and relative GSSG/GSH ratio was assessed according to the manufacturer’s instructions.
Oxiselecttm total glutathione gssg gsh assay kit
Manufactured by Cell Biolabs
Sourced in United States
The OxiSelectTM Total Glutathione (GSSG/GSH) Assay Kit is a colorimetric assay for quantitative measurement of total glutathione (GSH and GSSG) in biological samples. The kit utilizes a proprietary method to measure both reduced and oxidized glutathione levels.
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4 protocols using oxiselecttm total glutathione gssg gsh assay kit
1
Measuring Oxidative Stress in BON1 Cells
2
Quantification of Salivary Glutathione
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The concentration of total glutathione (the sum of GSH and GSSG) was assessed by the use of an OxiSelectTM Total Glutathione (GSSG/GSH) Assay Kit (Cell Biolabs, INC, San Diego, CA, USA) and an EL-800 automatic microplate reader (Bio-Tek Instruments, INC, Winooski, VT 05404, USA). The method is based on the formation of a color product in the tested material. Measurement of the resulting color was made at wavelength λ = 405 nm [25 (link)]. Glutathione concentration refers to a standard curve prepared from a GSSG standard solution, and it is expressed in µmol/l. The final values are expressed in µmol/mg of total saliva protein. The protein concentrations of the samples were determined on the basis of the bicinchoninic acid (BCA) method in accordance with the manufacturer’s (Sigma-Aldrich, St. Louis, MO 63118, USA) instructions, with bovine serum albumin (BSA) adopted as the standard.
3
Biochemical Investigations of Antioxidant and Oxidative Stress Markers in Type 2 Diabetes
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Six mL of fasting venous blood from 426 T2D patients and 136 healthy subjects was drawn for biochemical investigations of glutathione and reactive oxygen/nitrogen species using the Varioscan Flash microplate reader (Thermo Fisher Scientific, USA). Glutathione levels were measured using the OxiSelectTM Total Glutathione (GSSG/GSH) Assay Kit (Cell Biolabs, San Diego, CA, USA). The ROS/RNS levels were measured using the OxiSelectTM In Vitro ROS/RNS Assay Kit (Cell Biolabs, USA). The concentration of glucose, glycated hemoglobin, total cholesterol, high- and low-density lipoproteins, and triglycerides were assessed using the semi-automatic biochemical analyzer Clima MC-15 (RAL, Sevilla, Spain) and reagent kits from Diacon-DS (Moscow, Russia). The Cobas 6000 Roche Diagnostics (Basel, Switzerland) analyzer was used for measuring the plasma concentration of C-peptide by a competitive solid-phase chemiluminescent enzyme immunoassay.
4
Quantification of Plasma Oxidative Stress
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Plasma ROS levels were assessed in 426 T2D patients and 153 healthy volunteers, whereas GSH levels were assessed in 258 patients and 137 controls were recruited during the final phase of this study. The total GSH levels were determined by colorimetric assay using the OxiSelectTM Total Glutathione (GSSG/GSH) Assay Kit (Cell Biolabs, USA). The ROS levels were quantified by a fluorometric assay using the OxiSelectTM In Vitro ROS/RNS Assay Kit (Cell Biolabs, USA). The assay employs a proprietary quenched fluorogenic probe, dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ), which is a specific ROS/RNS probe. It was first primed with a quench removal reagent and subsequently stabilized in the highly reactive DCFH form. In this reactive state, ROS and RNS can react with DCFH, which is rapidly oxidized to the highly fluorescent 2′,7′-dichlorodihydrofluorescein (DCF). The standard curve of H2O2 was used to quantify the levels of free radicals such as ROS and RNS in plasma samples. Absorbance was measured on a microplate reader Varioscan Flash (Thermo Fisher Scientific, Waltham, MA, USA) at 405 nm and fluorescence at 480 nm excitation/530 nm emission.
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