The level of Glutathione Reductase (GR) activity, in hCM trated with H2O2 (Fig. 5 D), was measured by ELISA assay (ab83461, Abcam, Cambridge, UK) following the manufacturer’s instructions. Briefly, Post_EVs and H2O2 treated (1hr and 1hr respectively) cells were lysed by the addition of the lysis buffer provided in the kit and centrifuged at 14,000×g for 10 min. The GR in the supernatant reacts with DTNB to generate 2-nitro-5-thiobenzoic acid. This compound is yellow, and the sample's absorbance (405 nm) was measured by a microplate reader (TECAN). The results were normalized to total protein, measured by BCA (as previously described).
Ab83461
Manufactured by Abcam
Sourced in United Kingdom
Ab83461 is a primary antibody product manufactured by Abcam. It is designed for use in common research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab102530, by Abcam (4 mentions)
Ab238537, by Abcam (2 mentions)
Mbs726781, by MyBioSource (2 mentions)
90 mm diameter culture dish, by SPL Life Sciences (1 mentions)
Sunrisetm, by Tecan (1 mentions)
Mbs036924, by MyBioSource (1 mentions)
Ab83464, by Abcam (1 mentions)
Au400 chemistry analyzer, by Olympus (1 mentions)
7 protocols using ab83461
1
Glutathione Reductase Activity Measurement
2
Quantifying Glutathione Reductase Activity
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The activity of GR in plasma was determined using a specific assay kit(ab83461, Abcam, Cambridge, United Kingdom) [47 (link)]. In this assay, GSH is formed from GSSG by GR; then, GSH reacts with DTNB, and a 2-nitro-5-thiobenzoate anion (TNB2-) is generated [45 (link)]. The change in absorbance was measured at 405 nm. The kit reagents were dissolved as described in the “components and storage” section. The protocol recommends pretreating the samples. First, 5 μL of 3% H2O2 was added to 100 μL of each sample. The samples were incubated at 25°C for 5 min. Then, 5 μL of catalase was added to each sample, and we incubated the samples again at 25°C for another 5 min. After the pretreatment procedure, 50 μL of each pretreated sample was added to the sample wells. The standard curve was prepared as described in the manual, and 50 μL of diluted standard solution was added to each well. Measurements were performed according to a previous study [46 (link)]. GR activity is expressed in nmol/min/mL = mU/ml.
3
Intracellular GR Activity Assay
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Intracellular GR activity was examined using a commercially available assay kit (Cat. # ab83461; abcam, Cambridge, UK). HCT116 cells were plated in a 90 mm diameter culture dish (SPL Life Sciences co., Ltd.) at a density of 1 × 106 cells. Following 72 h of treatment with QUE (25 μM) in the absence or presence of 0.5 μM OXP and 3 μM SFN, the cells were detached and concentrated to a density of 1 × 106 cells in 25 μL of PBS. A 100 μL of cold assay buffer was added to the cell suspension and vortexed for lysis. The supernatant from the cell lysate was used for the assay as instructed by the manufacturer. After the reaction was completed, the absorbance at 405 nm was detected using a microplate reader (SunriseTM, Tecan Group Ltd.).
4
Spinal Cord GPx and GR Activity
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Protein allocations derived from spinal cord homogenate obtained at one and three dpi were used at 30-mg/well in duplicate. Commercial kits were obtained to analyze total GPx activity (ab102530; Abcam Co.), as well as total GR activity (ab83461; Abcam Co.). Baseline densities were obtained before starting the colorimetric assays, and kinetic readings were used to assess catalytic rates. Standard curves of obtained values were generated, and sample values were interpolated against best fit curves using Prism (version 9). For both activity assays at each time point, n = 4-5 mice were used per group before combining sexes. When sexes were combined, n = 8–10 mice/group were used.
5
Oxidative Stress Biomarker Assays
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Malondialdehyde (MDA; ab238537), glutathione peroxidase (GSH-Px; ab102530), and glutathione reductase (GSH-R; ab83461) ELISA kits were acquired from Abcam Inc. (Cambridge, UK). Superoxide dismutase (SOD; MBS036924) and catalase (MBS726781) ELISA kits were obtained from My BioSource (San Diego, CA, USA). All the procedures were performed in agreement with the manufacturer’s directions.
6
Oxidative Stress Biomarkers ELISA
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Malondialdehyde (MDA; ab238537), glutathione peroxidase (GSH-Px; ab102530) and glutathione reductase (GSH-R; ab83461) ELISA kits were acquired from Abcam Inc. (Cambridge, UK). Superoxide dismutase (SOD; MBS036924) and catalase (MBS726781) ELISA kits were obtained from My BioSource (San Diego, CA, USA). All the procedures were executed in agreement with the manufacturer’s directions.
7
Biochemical and Immune Profiles in Serum Samples
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The biochemical parameters and hormone levels influencing the levels of calcium (Ca), phosphorus (P), blood urea nitrogen (BUN), cholesterol (CHO), glucose (GLU), triglyceride (TG), albumin (ALB), progesterone (P4), estrogen (E), and cortisol hormone in serum samples were determined using an Olympus AU400 chemistry analyzer (Olympus Optical Co., Ltd., Tokyo, Japan) following the manufacturer’s instructions. Immune activities, including alternative complement hemolytic 50 (ACH50) activities, total immunoglobulin (total Ig), and lysozyme activities (LZY), were determined using a method previously described by Incharoen et al. [13 (link)] with slight modifications. SOD activities were also determined using a modification of the mentioned method [13 (link)], and the enzymatic activity was expressed as the percent inhibition rate. The catalase activity (CAT; EC1.11.1.6) was determined using a catalase assay kit (ab83464; Abcam, Cambridge, UK) according to the manufacturer’s standard procedures. The activity of glutathione peroxidase (GPx; EC.1.11.1.9) was determined using a GPx assay kit (ab102530; Abcam, Cambridge, UK) following the manufacturer’s standard protocols. The activity of glutathione reductase (GR; EC1.8.1.7) was measured using a commercially available assay kit (ab83461; Abcam, Cambridge, UK) according to the manufacturer’s standard processes.
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