At admission to the study and after the achievement of the weight loss goal, venous blood samples were collected and frozen at −20 °C for subsequent biochemical measurements. All subjects were studied as out-patients after a 12-h fast and overnight urine collection was performed immediately before blood sampling. Urine samples were added with the antioxidant 4-hydroxy-Tempo (1 mM) (Sigma Chemical Co., St. Louis, MO, USA) and stored at –20 °C until extraction.
4 hydroxy tempo
Manufactured by Merck Group
Sourced in United States, Germany
4-hydroxy-TEMPO is a stable free radical compound commonly used as a spin label in electron paramagnetic resonance (EPR) spectroscopy. It has a nitroxide functional group that provides a stable free radical center, allowing it to be used as a probe to study the dynamics and structure of biological systems. The core function of 4-hydroxy-TEMPO is to serve as a spin label for EPR spectroscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Methanol, by Merck Group (2 mentions)
Indomethacin, by Merck Group (2 mentions)
N acetyl l cysteine nac, by Merck Group (2 mentions)
2 deoxy d glucose, by Merck Group (2 mentions)
L glutamine 13c5, by Merck Group (2 mentions)
Tween 80, by Merck Group (2 mentions)
Trolox, by Merck Group (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Methylcellulose, by Merck Group (2 mentions)
Ferrostatin 1, by Merck Group (2 mentions)
Deferoxamine, by Merck Group (2 mentions)
D penicillamine, by Merck Group (2 mentions)
Sulfasalazine, by Merck Group (2 mentions)
6 aminonicotinamide, by Merck Group (2 mentions)
Bay 876, by Merck Group (2 mentions)
Epiandrosterone, by ChemScene (2 mentions)
L penicillamine, by Merck Group (2 mentions)
Benzyl alcohol, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
33 protocols using 4 hydroxy tempo
1
Blood and Urine Sampling Protocol
2
Culturing M1 Cells with CD9truc-EGFP
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The M1 cells with stable CD9truc‐EGFP expression were cultured as described previously.28 Cells were maintained in Dulbecco's Modified Eagle Medium, F‐12 Nutrient Mixture (Gibco, Sigma Aldrich) with 10% fetal bovine serum (FBS, Fisher Scientific) and 1% Penicillin–Streptomycin at 37°C in a 5% CO2 humidified incubator. For all cell‐conditioned medium analyses, the culture medium was changed to PC1 serum‐free medium (Lonza, No. 344018). CoCl2 (Sigma Aldrich), Roxadustat (Astatech), DCA (Sigma Aldrich), 4‐Hydroxy‐TEMPO (Sigma Aldrich) and 4‐nitrobenzoate (Sigma Aldrich) was dissolved in PC1‐serum‐free medium and added to a final concentration of 100 μM, 30 μM, 30 mM, 2 mM, and 2 mM, respectively. Pitavastatin (Santa Cruz) and Rotenone (Sigma Aldrich) were dissolved in dimethyl sulfoxide and added to a final concentration of 5 μM and 10 μM, respectively, and antimycin A (Sigma Aldrich) was dissolved in 99% ethanol and added to a final concentration of 1 μM. Dissolved drugs were added when cells reached 60–80% confluence in 6‐well plates, and cells and medium were harvested after 24 h incubation.
3
Synthesis and Characterization of Hybrid Biomaterials
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PEG (polyethylene glycol), urea, acetic acid, trimethoxy orthosilicate (TMOS), Chlorotrimethylsilane(TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy-TEMPO, benzoyl peroxide (BPO), HPLC grade acetonitrile (ACN), methanol, 2-propanol, and acetone were purchased from Sigma–Aldrich (St. Louis, MO, USA).
4
Tempol Infusion in Healthy Sheep
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In nonanesthetized healthy sheep, baseline measurements commenced 4 days after the second surgical procedure. In five healthy nonseptic sheep, animals received a renal arterial infusion of tempol (3 mg kg−1 h−1, 4‐Hydroxy‐TEMPO, Sigma‐Aldrich) for 4 h. Animals were allowed 24 h as a washout period and then received an IV infusion of tempol (30 mg kg−1 h−1) for 4 h. Arterial blood and renal venous blood were collected prior to tempol infusion and then at hourly intervals during the 4‐h continuous infusion of renal arterial and IVT infusion and then at 15 min, 45 min, and 2 h postinfusion to determine pharmacokinetic parameters (see Online Supplement for methodology).
5
Fabrication of Perovskite Solar Cells
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Acrylamide (purity ≥98.0%), dimethyl sulfoxide (DMSO, ≥99.7%), poly(ethylene glycol) diacrylate (PEGDA, Mn = 700), 4-hydroxy-TEMPO (97%), benzyl viologen dichloride (97%), LiTFSI (99.95% trace metals basis) and 1-Hydroxycyclohexyl phenyl ketone (99%) were purchased from Sigma-Aldrich (Seoul, Korea). Poly(sodium 4-styrene sulfonate) (PSSNa, Mw ~ 1,000,000, 20 wt% in H2O) solution was purchased from Tosoh Corporation (Tokyo, Japan). A PSSA solution was prepared by treating PSSNa with an ion-exchange resin (TRILITE UPRC100U, Samyang Corporation). MXene (Ti3C2TX, 3–5µm) was purchased from Invisible Corporation (Suwon, Korea). MXene was made by etching Ti3AlC2 powder with 40% HF solution for 72 h.
6
Tissue Expansion and Labeling Protocol
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For expansion of tissue, brain slices were pre-incubated with TREx gelation solution for 30 min on ice in the presence of 15 µg ml−1 4-hydroxy-TEMPO (Sigma-Aldrich, 176141) to delay premature gelation. To create the gelation chamber, tissue slices were laid out on a microscopy slide with four dabs of vacuum grease surrounding the slice. A GelMap coverslip was placed with the protein grid facing the tissue on top of the dabs of vacuum grease and pressed down ensuring contact with the tissue slice. The gelation chamber was filled with gelation solution from the side and transferred to 37 °C for 1 h. After gelation, the gel surrounding the tissue was trimmed and the sample was denatured for 3 h at 80 °C in disruption buffer containing 5% SDS, 200 mM NaCl and 50 mM Tris, pH 7.5. After disruption, gels were washed in PBS and either first processed for post-expansion labeling as described above or directly stained with 30 µg ml−1 NHS ester conjugated to ATTO488 (ATTO-TEC) for 2 h at RT. After staining, gels were washed and expanded in MQ.
7
Phenotyping and Antioxidant Treatment of Diabetic ADSCs
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ADSCs were obtained from the subcutaneous adipose tissues in the inguinal area of
10-week-old STZ-induced diabetic and control C57 mice as described previously21 (link). Isolated ADSCs were plated at 5×105 cells/cm2 in DMEM with
low glucose (5 mM). To determine the phenotype of the dADSCs, the ADSCs were washed with
phosphate-buffered saline (PBS) and incubated with phycoerythrin-conjugated anti-mouse
antibodies against CD11b, CD29, CD31, CD44, CD90.1, CD133, and major histocompatibility
complex II (MHC-II) for 25 min at 4°C in the dark. The cells were then washed with PBS and
collected for flow cytometry analysis (Beckman Coulter, Fullerton, CA, USA). Cultured
ADSCs were passaged when they reached 75–80% confluence. The initial confluent culture was
designated passage 0. Cultured dADSCs from passage 3 were treated with either a general
antioxidant, 4-Hydroxy-TEMPO, formally 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl
(TEMPO, Sigma-Aldrich, St Louis, MO, USA) (1 µM) or a mitochondrially targeted
antioxidant,
(2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium
chloride (mitoTEMPO, Sigma) (1 µM) for three passages and then used in experiments. The
nADSCs were not treated with TEMPO or mitoTEMPO during the experiments.
10-week-old STZ-induced diabetic and control C57 mice as described previously21 (link). Isolated ADSCs were plated at 5×105 cells/cm2 in DMEM with
low glucose (5 mM). To determine the phenotype of the dADSCs, the ADSCs were washed with
phosphate-buffered saline (PBS) and incubated with phycoerythrin-conjugated anti-mouse
antibodies against CD11b, CD29, CD31, CD44, CD90.1, CD133, and major histocompatibility
complex II (MHC-II) for 25 min at 4°C in the dark. The cells were then washed with PBS and
collected for flow cytometry analysis (Beckman Coulter, Fullerton, CA, USA). Cultured
ADSCs were passaged when they reached 75–80% confluence. The initial confluent culture was
designated passage 0. Cultured dADSCs from passage 3 were treated with either a general
antioxidant, 4-Hydroxy-TEMPO, formally 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl
(TEMPO, Sigma-Aldrich, St Louis, MO, USA) (1 µM) or a mitochondrially targeted
antioxidant,
(2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium
chloride (mitoTEMPO, Sigma) (1 µM) for three passages and then used in experiments. The
nADSCs were not treated with TEMPO or mitoTEMPO during the experiments.
8
Vascular Reactivity in High Glucose
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The experiments were performed in a KRB solution of the following composition (mM): NaCl 135, KCl 5, NaHCO3 20, glucose 10 (control) or 30 (HG), CaCl2 2.5, MgSO4 1.3, KH2PO4 1.2 and EDTA 0.026 in H2O. l -phenylephrine hydrochloride (Phe), acetylcholine chloride (Ach), SNP, Nω-Nitro-l -arginine methyl ester hydrochloride (L-NAME), indomethacin (indo), MGO solution, 4-hydroxy-tempo (Temp), l -ascorbic acid (AA), resveratrol, quercetin and dimethylsulfoxide (DMSO) were obtained from Sigma (St. Louis, MO). Stock solutions were made in water except for indo (ethanol). The final concentrations of vehicle solution in the organ bath never exceeded 0.1 %. Stock solutions of 100 mM of resveratrol and quercetin were made in DMSO, but were further diluted in distilled water (10 mM) before adding to the organ baths.
9
Investigating Cellular Responses to Chemical Treatments
10
Pharmacological Modulation of Endothelial Function
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Potassium chloride (KCl), PE, Nω-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor), ACh, sodium nitroprusside (SNP), 4-Hydroxy-TEMPO (tempol, a superoxide anion scavenger), indomethacin (a non-selective cyclooxygenase (COX) inhibitor), and methyl-β-cyclodextrin (CD, a cholesterol depleter which disassembles caveolae) were purchased from Sigma-Aldrich (St. Louis, MO, USA); losartan (an Ang II type 1 receptor antagonist) was from Cayman Chemical Company (Ann Arbor, MI, USA). Drug concentrations were expressed as final molar concentrations in the bath chamber. 10× radio-immunoprecipitation assay (RIPA) lysis buffer was from Millipore Corporate Headquarters (Temecula, CA, USA); bicinchoninic acid (BCA) protein assay kit and Pierce enhanced chemiluminescent (ECL) Western Blotting Substrate were from Thermo Fisher Scientific (Rockford, IL, USA); anti-p-eNOSThr495 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-eNOSSer1177 was from Abcam plc (Cambridge, UK); anti-eNOS, anti-adenosine monophosphate-activated protein kinase (anti-AMPK), anti-Cav-1, anti-β-actin, and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG were from BD Transduction Laboratorie (Lexington, KY, USA); HRP conjugated goat anti-rabbit IgG was from Cell Signaling Technology (Danvers, MA, USA).
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