Peripheral blood was collected in EDTA- or sodium heparin-containing tubes. PBMCs were isolated by Histopaque® density gradient centrifugation (Sigma-Aldrich, Germany) following the manufacturer’s instructions and were washed once (centrifuged at 235 ×g for 15 min) in PBS (Sigma-Aldrich, Germany). Pellets were re-suspended in RPMI-1640 medium, labeled with cell-specific, fluorescence-marked antibodies (CD4+ or CD8+ lymphocyte subsets were identified by binding of PerCP-conjugated monoclonal antibodies, all BD Heidelberg, Germany) and incubated with the dye Mercury-Orange (Sigma-Aldrich, Germany) for 5 min at room temperature. Intracellular GSH concentrations were determined by flow cytometry using BD FACSCalibur Flow Cytometer.
Mercury orange
Manufactured by Merck Group
Sourced in Germany, United States
The Mercury orange is a specialized laboratory equipment that is designed to measure the concentration of mercury in various samples. It utilizes advanced detection technology to provide accurate and reliable results. The core function of this product is to facilitate the quantification of mercury levels in environmental, industrial, or research applications.
Automatically generated - may contain errors
Lab products found in correlation
Cell proliferation kit 2 xtt, by Roche (3 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Ethanol, by Merck Group (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Histopaque density gradient centrifugation, by Merck Group (1 mentions)
Oxidized glutathione, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Glutathione reductase, by Merck Group (1 mentions)
Nicotinamide adenine dinucleotide phosphate, by Merck Group (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (1 mentions)
2 4 dinitrophenylhydrazine, by Merck Group (1 mentions)
2 vinylpyridine, by Merck Group (1 mentions)
10 protocols using mercury orange
1
Intracellular GSH Levels in PBMC
2
Oxidative Stress Biomarker Assay Protocol
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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), and L-glutamine and trypsin were purchased from Gibco (Grand Island, NY). Tert-butyl hydroperoxide (tBHP), 2,4-dinitrophenylhydrazine (DNPH), urea, oxidized glutathione (GSSG), nicotinamide adenine di-nucleotide phosphate (NADPH), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), 2-vinyl pyridine, glutathione reductase, ethyl acetate, Bradford reagent, mercury orange, and 2,7-dichlorofluorescein diacetate (DCF-DA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Trichloroacetic acid (TCA), sodium hydroxide (NaOH), 2-thiobarbituric acid (TBA), and ethanol were purchased from Merck (Darmstadt, Germany). Cell proliferation kit II (XTT) was purchased from Roche Diagnostics (Mannheim, Germany).
3
Glutathione Antioxidant Evaluation using Mercury Orange
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Reduced glutathione (GSH) evaluation, an antioxidant defense, was performed with the fluorescent compound mercury orange. This compound reacts with GSH and this reaction product emits red fluorescence when excited at 488 nm [22 (link),23 (link)]. A cell suspension of 106 cells was incubated for 15 min at room temperature in the dark with 40 mM of mercury orange (Sigma 83377). After washing the cells with PBS, fluorescence was determined at 485/20 nm excitation and 590/35 nm emission [23 (link)].
4
Mercury Orange Fluorescence Assay
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Cells were incubated with 10 μM of mercury orange (Sigma Aldrich, St. Louis, MO, USA) for 15 min in the dark at 37 °C. Samples were read with excitation and emission wavelengths of 485 and 590 nm, respectively, in the multi-well plate spectrophotometer.
5
Detecting Oxidative Stress in Muscle Sections
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Soon after sacrifice, the remaining quadriceps SMs of three mice per group were removed and snap frozen in OCT. Serial cryostat sections were obtained. Immediately after cutting, at least three sections were stained with the sulfhydryl-reactive dye Mercury Orange (MO; Sigma–Aldrich) as previously described [12 ]. Briefly, sections were incubated on ice in MO in cold 90% acetone at a final concentration of 25 μM. To confirm the specificity of MO binding to nonprotein thiols (including glutathione and cysteine), control sections were pretreated with 100 mM N-ethylmaleimide (Sigma–Aldrich) for 10 min to block thiol groups. For ROS staining, a corresponding number of sections from the same SM samples were fixed for 10 min in acetone, followed by incubation with 10 μM 2′,7′-dichlorofluorescein diacetate (H2DCFDA; Molecular Probes) for 30 min at 37 °C and a wash in PBS. Finally, confocal microscopy images were acquired by the Fluoview FV500 software.
6
Antioxidant Screening of Cell Culture Reagents
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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS) and trypsin were purchased from Gibco (Grand Island, NY, USA). Ethylenediaminetetraacetic acid (EDTA), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), diammonium salt (ABTS), horseradish peroxidase (HRP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric chloride, 2-deoxyribose, nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), mercury orange and 2,7-dichlorofluorescein diacetate (DCF-DA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Trichloroacetic acid (TCA) and 2-thiobarbituric acid (TBA) were purchased from Merck (Darmstadt, Germany) and potassium ferricyanide was obtained from AppliChem (Dresden, Germany). The cell proliferation kit II (XTT) was purchased from Roche Diagnostics (Mannheim, Germany).
7
Assessing Cellular Responses to Environmental Toxicants
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Mercury Orange (1-(4-chloromercuriophenylazo)-2-naphthol), Annexin V-FITC, DCFDA (2′,7′-dichlorofuorescein diacetate), JC-1 (1H-Benzimidazolium, 5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-, iodide), propidium iodide (PI), glyphosate (PESTANAL®, analytical standard), imidacloprid (PESTANAL®, analytical standard), and imazalil (PESTANAL®, analytical standard) were obtained from Sigma-Aldrich (Merck, Germany). Versene, trypsin-EDTA, Dulbecco’s Modified Eagle Medium (DMEM), streptomycin, penicillin, L-glutamine sodium pyruvate, foetal bovine serum (FBS), and DHPE-FITC (N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine) were obtained from Gibco (Alfagene, Invitrogen, Portugal). Alamar Blue® was purchased from Invitrogen, Life-Technologies (Porto, Portugal). Caco-2 and HepG2 cell lines were purchased from Cell Lines Service (CLS; Eppelheim, Germany).
8
Antioxidant Capacity Assays Protocol
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Folin Ciocalteu, sodium carbonate, gallic acid, ethanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), methanol, 2,2΄-Azino-bis-(3-ethyl-benzthiazoline-sulphonic acid) (ABTS), horseradish peroxidase enzyme (HRP), hydrogen peroxide (H2O2), 2,7-dichlorofluorescein diacatete (DCF-DA) and mercury orange, were purchased from Sigma Aldrich (St Louis, MO, USA). Dulbescco’s modified Eagle medium (DMED), fetal bovine serum (FBS) and phosphate buffered saline (PBS) of analytical grade were purchased from Gibco (UK). Cell proliferation kit II (XTT) was purchased from Roche Diagnostics (Mannheim, Germany).
9
Glutathione Quantification in Erythrocytes
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The content of reduced glutathione was determined using mercury orange. Four µl erythrocytes were mixed in 1 ml Ringer. From the resulting cell suspension 150 µl was centrifuged (1600 rpm for 3 mins at RT). Cells were stained with mercury orange in PBS (40 µM, Sigma Aldrich, Germany) and incubated for 3 min at 37°C, washed once and resuspended in 200 µl PBS. The fluorescence intensity was measured with flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 576 nm.
10
Glutathione Quantification by Mercury Orange
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The content of reduced glutathione was measured using mercury orange. After incubation, erythrocytes were washed in Ringer solution and then loaded with 40 µM mercury orange (Sigma-Aldrich, Hamburg, Germany) in PBS and incubated at 37°C for 3 mins. Afterwards, the samples were washed once and were finally resuspended in 200 µl PBS. The fluorescence intensity was measured in FL-2 channel by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 576 nm.
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