The levels of IL-6, TNF-α, MDA, and GSH were analyzed using commercial ELISA kits according to the manufacturer’s instructions (IL-6: R&D Systems, Minneapolis, MN, USA, DY406-05; GSH: Cayman, Ann Arbor, MI, USA, 703002; TNF-α: R&D Systems, Minneapolis, MN, USA, DY410-05; MDA: BioVision, Waltham, MA, USA, k739-100). The protein levels were confirmed in duplicate and measured using a VICTOR X4 Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).
Glutathione (gsh)
Manufactured by R&D Systems
Sourced in United States
The GSH is a laboratory equipment product that measures the levels of glutathione (GSH), a key antioxidant in cells. It provides a quantitative assessment of GSH concentrations in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione (gsh), by Cayman Chemical (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Victor x4 multimode plate reader, by PerkinElmer (1 mentions)
U 3010 spectrophotometer, by Hitachi (1 mentions)
Glutathione (gsh), by Fujifilm (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Tbars assay kit, by Cayman Chemical (1 mentions)
4 hne, by R&D Systems (1 mentions)
8 ohdg, by R&D Systems (1 mentions)
Mmp9 c 20 sc 6840, by Santa Cruz Biotechnology (1 mentions)
Ab7778, by Abcam (1 mentions)
Ab15537, by Abcam (1 mentions)
Ab41532, by Abcam (1 mentions)
Ab8880, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab46154, by Abcam (1 mentions)
5 protocols using glutathione (gsh)
1
Cytokine and Oxidative Stress Biomarkers
2
In Vitro Glo1 Assay Protocol
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In vitro Glo1 assay was performed as indicated in the protocol of R&D systems. The assay buffer used was 0.1 M sodium phosphate pH 7.0 (Nacalai Tesque). Glutathione, GSH (Wako) in distilled water (100 mM) and methylglyoxal, MG (Sigma) in assay buffer (100 mM) were prepared fresh. The substrate (MG-SG) mixture was generated by pre-incubating GSH and MG in the assay buffer at 25 C. Recombinant human glyoxalase 1, rhGlo1 (R&D Systems) or recombinant 6-His-mGLo1 in assay buffer was incubated either with DMSO or the compound. The substrate mixture was added to the enzyme and the change in absorbance at 240 nm was recorded for 3 min using Hitachi U-3010 Spectrophotometer. The final concentration of rhGlo1 and 6-His-mGlo1 were 9.5 nM and 19 nM, respectively. For the kinetic studies, various concentrations of the substrate (MG-SG) were prepared by varying the concentration of MG while maintaining the concentration of GSH. The substrate concentration was calculated using the equation: 3 mM = Kd = ½MG½GSH=½MGSG. The inhibitory constants, Ki (E) and K'i (ES) values were graphically estimated as previously described (Cornish-Bowden, 1974) .
3
Oxidative Stress Biomarkers in Ischemic Brain
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The levels of MDA (TBARS assay kit, Cayman Chemical), protein carbonylation (Abcam, ab126287, Cambridge, UK), GSH (R&D System, Minneapolis, MN, USA), and catalase expression (Thermo Fisher Scientific Inc. EIACATC, Carlsbad, CA, USA) in the ischemic and control brains were measured with commercially available kits according to the manufacturers’ instructions. The absorbance was read on a microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA).
4
Oxidative Stress Markers in BALF
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Tracheal intubation was conducted after anesthesia into the rats, followed by three lavages with PBS. Then BALF was collected and centrifuged, and the supernatant was stored at −80°C. The concentrations of GSH, GSSG, 8-OHdG, and 4-HNE (R&D Systems, Minneapolis, MN) were measured in BALF by DAS-ELISA, according to the ELISA kits manufacturer's instructions.
5
Quantifying Tissue Biomarkers in Samples
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Immunohistochemistry kits were purchased from Abcam (Cambridge, UK), and as the primary antibodies against Ki67 (ab16667), VEGF (ab46154), collagen III (ab7778), FGF (ab8880), TGF-β3 (ab15537) and S100A4 (ab41532) were used. α-SMA (CGA7-sc53015) and MMP-9 (C20-sc6840) were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA) and collagen I (orb312178) was purchased from Biorbyt (Cambridge, UK). The total protein content of samples was determined by the Biuret method (Katal Biotecnológica Ind. Com. Ltd.a., Minas Gerais, Brazil). ELISA kits for TNF-α and IL-10 and the antioxidant enzymes SOD, GSH, GR and MPO were purchased from R&D Systems (Minneapolis, MN, USA). Collagenase 1.2 IU (refuse drug) was purchased from pharmaceutical companies.
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