Superoxide production in BAECs was determined by electron spin resonance (ESR) (eScan, Bruker, Billerica, MA, United States) as we previously published (23 (link)–37 (link)). After treatment, cells were collected in cold modified Krebs/HEPES (KHB) buffer (99 mmol/l of NaCl, 4.69 mmol/l of KCl, 1.03 mmol/l of KH2PO4, 2.50 mmol/l of CaCl2, 1.20 mmol/l of MgSO4, 25.0 mmol/l of NaHCO3, 5.6 mmol/l of glucose, and 20.0 mmol/l of Na-HEPES, pH 7.35). The cell suspension was mixed with superoxide-specific spin trap CMH (1 mmol/l, #ALX-430-117-M250, Enzo Life Sciences, Farmingdale, NY, United States) in nitrogen gas bubbled KHB buffer containing diethyldithiocarbamic acid (5 μmol/l, #D3506, MilliporeSigma, St. Louis, MO, United States) and deferoxamine (25 μmol/l, #D9533, MilliporeSigma, St. Louis, MO, United States). Then, the cell mixture was loaded in glass capillaries and analyzed immediately by ESR. Superoxide signal was measured in the presence or absence of polyethylene glycol-superoxide dismutase (PEG-SOD) (20 U/ml, #S9549, MilliporeSigma, St. Louis, MO, United States). The PEG-SOD inhabitable superoxide signal was calculated and normalized to protein concentrations. The ESR settings used were: center field 3,480.00 G; sweep width 9.00 G; microwave frequency 9.79 GHz; microwave power 21.02 mW; modulation amplitude 2.47 G; 512 points of resolution; and receiver gain 1,000.
D9533
Manufactured by Merck Group
Sourced in United States
D9533 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of D9533 is to provide a reliable and consistent platform for various laboratory tasks and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Escan, by Bruker (3 mentions)
Alx 430 117 m250, by Enzo Life Sciences (3 mentions)
D3506, by Merck Group (3 mentions)
S9549, by Merck Group (3 mentions)
F3388, by Merck Group (2 mentions)
Cell counting kit 8 (cck8), by Vazyme (1 mentions)
Hy 15763, by MedChemExpress (1 mentions)
S1026, by Selleck Chemicals (1 mentions)
Male ob ob mice, by Jackson ImmunoResearch (1 mentions)
Desferroxamine, by Merck Group (1 mentions)
Sml0583, by Merck Group (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Iron dextran, by Merck Group (1 mentions)
F8633, by Merck Group (1 mentions)
App ps1 double transgenic mice, by Jackson ImmunoResearch (1 mentions)
13 protocols using d9533
1
Measurement of Superoxide Production in BAECs
2
Measuring Endothelial Superoxide Production
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Confluent BAECs were starved overnight in M199 medium containing 5% FBS, and incubated with 10% of e-CSE for 24 h. Superoxide production was measured using electron spin resonance (ESR) (eScan, Bruker, Billerica, MA, USA) as we previously described [15 (link),18 (link),19 (link)]. Briefly, BAECs were washed, and cell pellets collected in cold KHB buffer. Superoxide-specific spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH, 1 mmol/L, ALX-430-117-M250, Enzo Life Sciences, Framingdale, NY, USA) was prepared in nitrogen gas bubbled KHB buffer containing diethyldithiocarbamic acid (5 μmol/L, D3506, MilliporeSigma, St. Louis, MO, USA) and deferoxamine (25 μmol/L; D9533, MilliporeSigma, St. Louis, MO, USA). Resuspended cells were mixed with spin trap in the presence of water (baseline) or PEG-SOD (polyethylene glycol-superoxide dismutase; 20 U/mL; S9549, MilliporeSigma, St. Louis, MO, USA), and loaded immediately into ESR spectrometer. The PEG-SOD inhibitable signals were used to calculate superoxide production and normalized to protein concentrations.
3
Measuring Superoxide Production in Endothelial Cells
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Superoxide production in BAECs and HAECs was determined by ESR (eScan, Bruker) as we previously published [[35] (link), [36] (link), [37] (link), [38] (link), [39] (link),[45] (link), [46] , [47] (link), [48] (link), [49] (link), [50] (link), [51] (link)]. After indicated treatment protocols, BAECs or HAECs were collected in cold modified Krebs/HEPES (KHB) buffer (99 mmol/L of NaCl, 4.69 mmol/L of KCl, 1.03 mmol/L of KH2PO4, 2.50 mmol/L of CaCl2, 1.20 mmol/L of MgSO4, 25.0 mmol/L of NaHCO3, 5.6 mmol/L of glucose, and 20.0 mmol/L of Na-HEPES, pH 7.35). Cell suspension was mixed with superoxide-specific spin trap CMH (1 mmol/L, #ALX-430-117-M250, Enzo Life Sciences) in nitrogen gas bubbled KHB buffer containing diethyldithiocarbamic acid (5 μmol/L, #D3506, MilliporeSigma) and deferoxamine (25 μmol/L, #D9533, MilliporeSigma). Then the cell mixture was loaded in glass capillaries and analyzed immediately by ESR. Superoxide signal was measured in the presence or absence of polyethylene glycol-superoxide dismutase (PEG-SOD, 20 U/mL, #S9549, MilliporeSigma). The PEG-SOD inhabitable superoxide signal was calculated and normalized to protein concentrations. The ESR settings used were as follows: center field 3477 or 3480 G; sweep width 9.00 G; microwave frequency 9.79 GHz; microwave power 21.02 mW; modulation amplitude 2.47 G; 512 points of resolution; and receiver gain 1000.
4
Erastin, Vitamin C, and Ferroptosis
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Cells (3 × 103) were seeded into a 96-well culture plate and treated with erastin (HY-15763, MedChemExpress) and/or vitamin C with or without deferoxamine (DFO; D9533, Sigma–Aldrich) for 24 h. To assess the efficiency of cell proliferation, Cell Counting Kit-8 (CCK8, A311-01/02, Vazyme) was used according to the manufacturer's protocol. The absorbance value (OD) was measured with a microplate reader at 450 nm.
5
Macrophage Response to Echinomycin and Deferoxamine
6
Intra-Tibial Injection for Bone Marrow Transplantation
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The intra‐tibial injection procedure was applied according to the bone marrow transplantation protocol described previously.31 , 68 Briefly, the tibia was pushed up and slightly rotated to fix the knee at ~90°. A 30 G needle was first inserted through the patellar tendon and drilled parallel along the long axis of the tibia to enter the proximal bone marrow cavity. Then, the needle was withdrawn, and a microsyringe (80401, Hamilton, USA) was carefully inserted into the drilled tunnel, and 10 μl of 100 μg/ml recombinant PDGF‐BB protein (315‐18‐50, Peprotech, USA), 50 mM IM (S1026, Selleck, USA), or equivalent volumes of PBS as placebo were slowly injected. Reagents were injected once every week and continued for 4 weeks. For irradiated mice, intra‐tibial injection was performed on days 0, 7, 14 and 21 after irradiation.
For intra‐peritoneal injection, DFM (D9533, Sigma‐Aldrich, USA) and IM were administered every other day for 21 consecutive days at doses of 100 and 70 mg/kg, respectively. For irradiated mice, the first injection was arranged on Day 7 after irradiation.
For intra‐peritoneal injection, DFM (D9533, Sigma‐Aldrich, USA) and IM were administered every other day for 21 consecutive days at doses of 100 and 70 mg/kg, respectively. For irradiated mice, the first injection was arranged on Day 7 after irradiation.
7
Deferoxamine Treatment in Obese Mice
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Male ob/ob mice (aged 24 weeks, Jackson Laboratory) were randomly divided into groups: vehicle and DFO (8 mice per group). As described in previous reports, mice in the DFO group were given DFO (Sigma D9533, 100 mg/kg body weight, dissolved in saline, intraperitoneally once a day (i.p.q.d.)) for 15 days (10 (link)). Mice in the vehicle group were treated with the vehicle control. Obese mice and age-matched WT (C56BL/6J) mice were housed in a temperature-controlled (22 ± 2°C) environment on a 12-h light/dark cycle with free access to food and water. The general health and body weights of the animals were monitored for the duration of the experiment. After the treatment, the white adipose tissues of the epididymal fat were rapidly collected with the animals under anesthesia, and bisected halves were fixed in 4% paraformaldehyde in phosphate-buffered saline or snap-frozen in liquid nitrogen and stored at −80°C for future assessment. Experimental procedures were performed in accordance with the National Institutes of Health guidelines and were approved by the Laboratory Animal Ethical Committee of Northeastern University.
8
Isolation and Treatment of Cardiomyocytes
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Adult primary cardiomyocytes were isolated from eight week old C57BL/6 mice. Hearts were cannulated and mounted on a langendorff apparatus, then perfused using a liberase solution for 10 min. After filtration through a 400 µm gauze, cells were cultured in MEM medium containing Hanks salts, L-glutamine and antibiotics. Within 2 hr of cardiomyocyte culture, supernatants were replaced with fresh medium containing 10% Fetal calf serum, with 0.5 mmol/L ferric citrate (FAC) (F3388, Sigma Aldrich) or 100 µmol/L desferroxamine (D9533, Sigma Aldrich) for 8 hr. The Furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK) (N1505, Bachem) was added at a concentration of 50 µmol/L for the duration of DFO and FAC treatment.
9
Fluoroquinolone Toxicity in HEK293 Cells
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10 mm stock solutions of CIPRO, ENRO, and NOR (Sigma, 17850, 17849, and N9890, respectively) were prepared by dissolving FQs in 0.01 n HCl. HEK293 cells were cultured in 10-cm dishes to 70% confluence prior to treatment with FQs at the indicated final concentrations for the indicated times. In some experiments, cells were treated with either 100 μm DFO (Sigma, D9533) or CoCl2 for 4 h. An equal volume of 0.01 n HCl (NT) was added to a separate dish of cells as negative control. In co-treatment experiments, cells were first treated with CIPRO for 30 min followed by addition of the indicated concentrations of ferric citrate (Sigma, F3388) for 4 h. Cells viability was determined using trypan blue dye exclusion.
10
Cytotoxicity Assay of Anti-Cancer Drugs
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Cell viability was detected by Cell Counting Kit-8 (CK04, Dojindo, Kumamoto, Japan). Cells seeded at 5000 cells/well density 96-well plates, 24 h later cells were treated with cisplatin, paclitaxel, or BMS-1 (HY-17394, HY-B0015, HY-19991, MCE, China) for 24 h. Treatment drug concentrations were as follows: cisplatin at doses of 0, 5, 10, 20, 40, 80 µM, paclitaxel at doses of 0, 5, 10, 20, 40, 80 nM, BMS-1 at doses of 0, 2.5, 5, 10, 20, 40 µM. For Fer-1(SML0583, Sigma-Aldrich, United States) or DFO(D9533, Sigma-Aldrich, United States) treament after transfection of HIC1 siRNAs, after transfected with siRNAs for 48 h, cells were treated 2uM Fer-1 or 20 uM DFO for 24 h. Then, 10 µL CCK-8 was added to each well of the 96-well plate and co-incubated with cells for 1 h. Then, the OD values at 450 nm were detected.
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