The antibodies to CA9, p-RelA, RelA, p-IKKβ, IKKβ, p-IκBα, IκBα, LDH, HIF1α, SQSTM1, cleaved-caspase-3, cleaved-PARP1, NRF2, and actin were obtained from Cell Signaling Technology. The antibodies to GPX4 and CA9 were obtained from Abcam. The antibodies to LC3, MLKL, RIPK3, OPRL1, OPRM1, and HMGB1 were obtained from NOVUS. Desferrioxamine, β-mercaptoethanol, N-acetyl-l-cysteine, N-Acetyl-l-alaine, hydrogen peroxide solution, gemcitabine hydrochloride, sulfasalazine, cobalt chloride, cycloheximide, tocopherol, necrosulfonamide, hydroxychloroquine sulfate, EDTA, cytochalasin B, cytochalasin D, paclitaxel, crystal violet, protease inhibitor cocktail, Z-VAD-FMK, TNF, staurosporine, cycloheximide, and lipopolysaccharides were obtained from Sigma-Aldrich. Erastin, ferrostatin-1, lapatinib, JTC801, and compound libraries were obtained from Selleck Chemicals. BANORL24, SB612111, UFP101, and Trap101 were obtained from Tocris Bioscience.
Desferrioxamine
Manufactured by Merck Group
Sourced in United States
Desferrioxamine is a pharmaceutical compound used as a chelating agent. It is primarily utilized in the treatment of iron overload conditions, particularly in patients who have received frequent blood transfusions. Desferrioxamine binds to excess iron in the body, facilitating its removal through urine and feces.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Necrosulfonamide, by Merck Group (2 mentions)
Erastin, by Selleck Chemicals (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
N acetyl l cysteine, by Merck Group (2 mentions)
Bafilomycin, by Thermo Fisher Scientific (2 mentions)
Paraquat, by Merck Group (2 mentions)
Ascorbate, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Cobalt chloride, by Merck Group (1 mentions)
Tocopherol, by Merck Group (1 mentions)
Hydroxychloroquine sulfate, by Merck Group (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Lapatinib, by Selleck Chemicals (1 mentions)
Crystal violet, by Merck Group (1 mentions)
P rela, by Cell Signaling Technology (1 mentions)
Sqstm1, by Cell Signaling Technology (1 mentions)
Hmgb1, by Novus Biologicals (1 mentions)
Jtc 801, by Selleck Chemicals (1 mentions)
Ufp101, by Bio-Techne (1 mentions)
22 protocols using desferrioxamine
1
Comprehensive Immunoblot Analysis
2
Cell Treatment with Bafilomycin, DFO, and Paraquat
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Bafilomycin (Fisher Scientific) was dissolved in DMSO, while desferrioxamine (DFO) (Sigma) and paraquat (Sigma) were dissolved in distilled water. An equal volume of vehicle was used as a control for all experiments. 6 × 104 cells were seeded into each well of 12-well plate and drug treatments were initiated 24 hours later. For Bafilomycin and DFO treatment, cells were treated with 5-10 nM Bafilomycin or 8 mM DFO for 48 hours. For paraquat treatment, cells were treated with 200 μg/mL paraquat for a total of 96 hours. Media was changed 24 hours after the beginning of the paraquat treatment and cells were split 1:2 into a new 12-well plate 48 hours after drug addition.
3
Particle Matter Exposure Assessment
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ALIs or 80–90% confluent monolayers were challenged (into the apical compartment for ALI cultures) with PM stock suspensions diluted in serum-free/protein-free medium (see Supplementary methods ), supplemented with desferrioxamine (DFX) or N-acetylcysteine (NAC) (both Sigma-Aldrich) where indicated. All ALI culture challenges were performed in 6.5 mm diameter, 0.4-µm pore size collagen-coated polyester Transwell inserts, placed into wells of standard 24-well cell culture plates. All monolayer challenges were performed in standard collagen-coated 24-well plates, except for 2,7-dichlorofluorescein (DCF) experiments, which were performed in equivalent collagen-coated 96-well plates. Volumes used were 75 µl for Transwell (300 µl medium in basolateral compartment) and 96-well assays, and 400 µl for 24-well plates. The resultant supernatants were centrifuged at 16 000 × g for 10 min at 4°C before storage at −80°C.
4
Hypoxia and Iron Chelation Effects on Nestin
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Cells were plated at 5,000 cells/cm2 in 12-well plates and 24 h later media was replaced with pre-conditioned media from each oxygen tension (21% or 1%). For ambient (21%) oxygen tension, cells were then cultured in a standard humidified incubator at 37°C with 5% CO2. For 1% oxygen tension experiments, cells were cultured in humidified incubators at 37°C with 5% CO2 with oxygen tension reduced to 1% using supplemental nitrogen (Heracell 150, Thermo Fisher USA) for the indicated time. A subset of cells were treated with 100 uM desferrioxamine (Sigma, USA), an iron chelator known to stabilize HIF-1α under normoxic conditions. In addition, nestin expression was analyzed after treatment with 10 μM vascular endothelial growth factor (VEGF) inhibitor CBO-P11 (EMD Millipore).
5
Siderophore Acquisition Compounds
6
Escherichia coli K-12 Strain Construction
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Escherichia coli K-12 strains used in this study were constructed from MC4100 (53 (link)) and are listed in Table 1 . Lysogeny broth (LB) was prepared using LB broth EZMix powder (Lennox). LB agar (LBA) medium contained LB plus 1.5% agar (Becton Dickinson). ONPG (2-ortho-nitrophenyl-β-d -galactopyranoside) was purchased from Acros. Diethylenetriaminepentaacetic acid (DTPA) and desferrioxamine were obtained from Sigma-Aldrich. All other chemicals were of analytical grade. The growth medium was supplemented with ampicillin (50 μg/ml), chloramphenicol (12.5 μg/ml), kanamycin (25 μg/ml), or tetracycline (10 μg/ml) when necessary. To induce plasmid-borne gene expression, 0.2% l -arabinose or 0.4 mM IPTG (isopropyl-β-d -thiogalactopyranoside) was added to the medium.
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7
Analytical Characterization of Therapeutic Protein Solution
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DTPA, EDTA, glutathione (GSH), quercetin, coumarine-3-carboxylic acid (CCA), 7-(diethylamino)coumarin-3-carbohydrazide (CHH), desferrioxamine, ascorbate, potassium phosphate (KH2PO4), and multi-compendial grade polysorbate 20 (PS20) and PS80 were from Sigma-Aldrich (Merck KgaA, Darmstadt, Germany). Copper(II) chloride and iron(III) chloride were from Acros Organics (Thermo Fischer Scientifics, Waltham, MA, USA). Ultra-gradient HPLC grade acetonitrile was from J.T. Baker (Avantor Performance Materials, Radnor, PA, USA). Liquid chromatography-MS grade methanol was from Merck KgaA (Darmstadt, Germany). Ultrapure water was from a Millipore Milli-Q water system (Merck KgaA, Darmstadt, Germany). The therapeutic protein solution was provided by Lek d.d. (Mengeš, Slovenia).
8
Chelation Compounds for Cell Culture
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Desferrioxamine (>99%), deferiprone (1, 2-dimethyl, 3-hydroxy, pyrid-4-one) and 100% cell culture grade DMSO were obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON). Desferasirox (4-[3, 5-bis (2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid) was synthesized in house according to established protocols [32] . The analysis and characterization of the final product are shown in supplementary materials (Figures S1 and S2 in File S1 ). Aquacalm was obtained from Syndel Laboratories Ltd. (British Columbia, Canada).
9
Measuring Mitochondrial Superoxide in Lung
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To measure mitochondrial superoxide levels in lung tissue, we performed electron paramagnetic resonance (EPR) measurements. Mouse lung tissues was pulverized to form fine tissue powder. Then 4–6 mg of tissue powder was dissolved in 300 μL of EPR buffer (PBS supplemented with 5 uM diethyldithiocarbamate and 25uM desferrioxamine; Sigma-Aldrich) at 0 °C in a temperature-controlled cooling shaker (UltraCruz; Santa Cruz). The dissolved powder (75 μL) was separated between two 1.5 ml tubes containing spin probe CMH (5 mg/ml) only and CMH plus the mitochondria respiratory chain inhibitor, Rotenone (1 mM) and incubated for 2 min at 0 °C. 30 μl of tissue suspension from each tube was transferred to a glass capillary at room temperature. Generation of superoxide in capillary took place for 30 min at room temperature. Superoxide radical generation was measured by reaction with spin probe CMH at room temperature in Magnettech M100 instrument (Magnettech, Germany). EPR spectra were analyzed for amplitude using ANALYSIS 2.0 software (Magnettech). To quantitate the amount of superoxide per milligram of protein, we also performed a standard reaction of the superoxide-generating enzyme xanthine oxidase in the presence of xanthine and CMH. Final superoxide generation rate was expressed as pmol/min/mg of tissue.
10
Cell Treatment with Bafilomycin, DFO, and Paraquat
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