Erastin (Selleck, Houston, TX, USA), RSL3 (Cayman Chemicals, Ann Arbor, MI, USA), ML162 (Cayman Chemicals), dimethyl fumarate (DMF; Santa Cruz Biotechnology, Dallas, TX, USA), Tert‐butyl Hydroperoxide (TBHP; Santa Cruz), 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB; Santa Cruz), crystal violet (Santa Cruz), monosodium glutamate (TCI, Portland, OR, USA), reduced glutathione (GSH; Millipore‐Sigma, St. Louis, MO, USA) and deferoxamine (DFO, Millipore‐Sigma) were purchased from the indicated companies.
Deferoxamine dfo
Manufactured by Merck Group
Sourced in United States
Deferoxamine (DFO) is a laboratory chelating agent used for the removal and treatment of excess iron and other metal ions in biological samples. It functions by forming stable complexes with metal ions, facilitating their removal and excretion from the body. Deferoxamine is a widely used tool in analytical and research applications that involve the study of metal-related processes and toxicities.
Automatically generated - may contain errors
Lab products found in correlation
Ferric ammonium citrate, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Saos 2, by American Type Culture Collection (4 mentions)
Erastin, by Merck Group (3 mentions)
Erastin, by Selleck Chemicals (2 mentions)
Cocl2, by Merck Group (2 mentions)
3 methyladenine 3 ma, by Merck Group (2 mentions)
Z vad fmk, by Merck Group (2 mentions)
Ferrostatin 1 fer 1, by Merck Group (2 mentions)
Cytochalasin d, by Merck Group (2 mentions)
Iron dextran, by Merck Group (2 mentions)
Nec 1, by Abcam (2 mentions)
Fer 1, by Merck Group (2 mentions)
Ferrostatin 1, by Merck Group (2 mentions)
Z vad fmk, by Enzo Life Sciences (2 mentions)
Ferrostatin 1 ferr 1, by Merck Group (2 mentions)
Mg132, by Merck Group (2 mentions)
Erastin, by MedChemExpress (2 mentions)
Hemin, by Frontier Scientific (2 mentions)
50 protocols using deferoxamine dfo
1
Reagent Procurement for Cell Metabolism
2
Hypoxia Induction Methods in LN229 and HeLa Cells
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For hypoxia experiments in LN229 cells, CoCl2 was used to mimic hypoxia conditions [27 (link)]. The cells were plated 2 days prior to the initiation of treatment, and the CoCl2 stock solution (100X in water) was added to the culture medium. At 16 h after treatment with CoCl2, the cell samples were collected. For hypoxia experiments for Hela cells, the cells were plated 1 day prior to the initiation of treatment. Exposure of cells to hypoxia was carried out in the PROOX C-Chamber with oxygen (O2) and carbon dioxide (CO2) levels modulated by the PROOX Model C21 Controller (BioSpherix, Lacona, NY, USA). The cells were exposed to 4% O2 for 10 h (moderate hypoxia) or exposed to six cycles of 1% O2 for 1 h followed by normoxia for 10 min, a combined total of 7 h of exposure (intermittent hypoxia). M4N stock solution was made in 100% dimethyl sulfoxide (DMSO). The final concentration of DMSO in the culture medium was 1.0%. Deferoxamine (DFO; Millipore Sigma) was added to the cultures as a 100X stock prepared in water (15 mM final concentration).
3
Ferroptosis Inhibition Mitigates IVH Injury
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Three or more independent experiments were performed for all experiments. Animals and cell cultures for each group were randomized with the website www.randomization.com . Treatment, data collection, and data analyses were blinded by using different investigators or by masking sample labels.
For in vitro experiments, we tested different dosages of Hemin (0, 10, 50, or 100 μM) and RSL3 (0, 2, 4, 8, 16, or 32 μM) at 12 and 24 h respectively. We added 2 μM Ferrostatin-1 (Fer-1, S7243, Selleck), 100 μM Deferoxamine (DFO, Y0001937, Sigma), 100 μM Necrostatin-1 (Nec-1, N9037, Sigma), 1 mM 3-Methyladenine (3-MA, HY-19312, MCE, USA), or 10 μM Q-VD-OPh (SML0063, Sigma) at the same time with Hemin or RSL3 based on previously published work [21 (link), 48 (link)]. We treated cells with 20 μM t-BOOH (458139, Sigma, USA) for 12 h, with or without Fer-1 or 0.5 mM GSH (G4251, Sigma, USA) at the same time. Cells were collected for PI staining, TUNEL staining, immunofluorescence staining, flow cytometry, GPx4 activity assay, RT-qPCR, and RNA-seq.
For in vivo experiments, Fer-1 (2 mg/kg) or vehicle (0.1% DMSO (D1418, Sigma), 2.5% PEG300 (202371, Sigma) and 0.25% Tween80 (P1754, Sigma) in saline) was injected daily (i.p.) for the initial 7 days beginning 2 h after IVH. The assessment included behavior tests, neuroimaging, histology, and TEM.
For in vitro experiments, we tested different dosages of Hemin (0, 10, 50, or 100 μM) and RSL3 (0, 2, 4, 8, 16, or 32 μM) at 12 and 24 h respectively. We added 2 μM Ferrostatin-1 (Fer-1, S7243, Selleck), 100 μM Deferoxamine (DFO, Y0001937, Sigma), 100 μM Necrostatin-1 (Nec-1, N9037, Sigma), 1 mM 3-Methyladenine (3-MA, HY-19312, MCE, USA), or 10 μM Q-VD-OPh (SML0063, Sigma) at the same time with Hemin or RSL3 based on previously published work [21 (link), 48 (link)]. We treated cells with 20 μM t-BOOH (458139, Sigma, USA) for 12 h, with or without Fer-1 or 0.5 mM GSH (G4251, Sigma, USA) at the same time. Cells were collected for PI staining, TUNEL staining, immunofluorescence staining, flow cytometry, GPx4 activity assay, RT-qPCR, and RNA-seq.
For in vivo experiments, Fer-1 (2 mg/kg) or vehicle (0.1% DMSO (D1418, Sigma), 2.5% PEG300 (202371, Sigma) and 0.25% Tween80 (P1754, Sigma) in saline) was injected daily (i.p.) for the initial 7 days beginning 2 h after IVH. The assessment included behavior tests, neuroimaging, histology, and TEM.
4
Establishing RPE1 Cell Lines for Hypoxia Modeling
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Human retinal pigment epithelium cell line (RPE1) was maintained in a 1:1 dilution of DMEM-Ham’s F-12 medium plus 10% FBS. RPE1 cells stably expressing pBabe or HPV-16 E7 were established by retrovirus-mediated infection using the pBabe-puro-based retroviral construct. Cells were selected with 10.5 μg/ml puromycin for 3 to 6 days. After the infected cells were pooled and expanded, they were maintained in puromycin (6.5 μg/ml) and used within 15 passages. The HPV-16 positive human cervical cancer cell line CaSki was purchased from American Type Culture Collection (ATCC). CaSki cells were cultured in DMEM supplemented with 10% FBS and antibiotics.
Deferoxamine (DFO, Sigma) and CoCl2 (Sigma) were used to mimic hypoxia at a dose of 200 μM and 500 μM, respectively.
Deferoxamine (DFO, Sigma) and CoCl2 (Sigma) were used to mimic hypoxia at a dose of 200 μM and 500 μM, respectively.
5
Programmed Cell Death Inhibitor Assay
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Caspase inhibitor Z-VAD-FMK (Sigma-Aldrich, United States), the autophagy inhibitor 3-Methyladenine (3-MA, Sigma-Aldrich, United States), the necroptosis inhibitorNecrostatin-1 (Nec-1,Sigma-Aldrich, United States), the ferroptosis inhibitor Ferrostatin-1 (Fer-1, Sigma-Aldrich, United States) and deferoxamine (DFO, Sigma-Aldrich, United States) were used in inhibitor investigations.
6
Hypoxia and Radiation Exposure of MDA-MB-231 Cells
7
Ferroptosis Induction and Measurement
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Erastin, lapatinib, ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1) and Ras-selective lethal small molecule 3 (RSL3) were obtained from Selleck Chemicals (Houston, TX, United States). Glutamate (Glu), propidium iodide (PI), Hoechst 33342, KA and deferoxamine (DFO) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Dulbecco’s modified Eagle’s medium (DMEM), Hank’s Balanced Salt Solution (HBSS) and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NY, United States). MDA Assay Kit and GSH and GSSG Assay Kit were purchased from Beyotime (Shanghai, China).
8
Zebrafish Mutant Maintenance and Bacterial Infection
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Adult zebrafish (Danio rerio) AB and embryos were maintained according to standard procedures at 28 °C [23 ]. A 10-h light/14-h dark day light cycle was used. The tp53zdf1/+ (AB) fish (Zebrafish International Resource Center, ZIRC) were in-crossed according to procedures reported by Berghmans et al. [14 (link)]. The genotype of the progeny was determined by restriction fragment length polymorphism (RFLP) (ZIRC) of the tail clip genomic DNA. The zdf1 allele harbors a single T-to-A point mutation in exon 7 that changes Met to Lys at residue 214 which gives the genotype tp53M214K. The zebrafish tp53M214K mutation is an ortholog to the human tp53M246K (c.737T > A; p.M246K; exon 7). Only tp53M214K/M214K were used in this study. The study was approved by the Institutional Animal Care and Use Committee (no. 10476). E. coli DH5a (Yeastern Biotech, Taiwan) and Vibrio vulnificus YJ016 [13 (link)] were grown in shaking Luria-Bertani (LB) broth at 37 °C. The S. iniae wild-type strain 9117 [24 (link)] was cultured at 37 °C in Todd-Hewitt medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.2% yeast extract (BD Biosciences, San Jose, CA, USA) and 2% proteose peptone (Sigma-Aldrich). Azacitidine (Vidaza®) was from Celgene (Summit, NJ, USA). Cytochalasin D, iron dextran and deferoxamine (DFO) were from Sigma-Aldrich.
9
Cell Death Pathway Inhibitors in NSCLC
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NucLight Red transduced NCI-H2228 and A549.R2 cells were seeded in a 96-well plate. After overnight incubation, cells were preincubated with the desired cell death pathway inhibitors for 1 h (1 μM ferrostatin-1 (Fer1, Sigma-Aldrich), 100 μM alpha-tocopherol (αToco, Sigma-Aldrich), 10 μM Z-VAD-FMK (Bachem, Bubendorf, Switzerland), and 10 μM necrostatin-1 s (Nec-1s, Abcam, Cambridge, UK) or 4 h (100 μM Deferoxamine, DFO, Sigma-Aldrich). Then, NCI-H2228 cells were treated with 5 μM AF, 30 μM olaparib, and their combination, and A549.R2 cells were treated with 6.5 μM AF, 30 μM olaparib, and theirs combination in the presence of the IncuCyte Cytotox Green Reagent (50 nM, Sartorius) or Caspase-3/7 Green Apoptosis Reagent (2.5 μM, Sartorius). Afterward, the plate was transferred to the temperature- and CO2-controlled IncuCyte ZOOM for 72 h. Cell death was monitored by pictures (10× magnification) that were taken every 24 h to limit phototoxicity. For analysis, green object count (1/mm2), red object count (1/mm2), green-red overlapping object count (1/mm2) and Caspase-3/7 Green GCU × μm2/image were determined with the IncuCyte basic analyzer. The percentage of cell death and Caspase-3/7 positive cells were calculated with the formula: green object count/(red object count − overlapping object count) × 100.
10
High-Throughput Cell Viability Assays
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MTS assays (CellTiter; Promega) were conducted as described previously (23 (link), 26 ). The Ramos, SUDHL4, Raji, Daudi, Namalwa, SUDHL6, and Jurkat cells were plated at a density of 2 × 105 cells/ml and cultured for 72 h prior to assay. SUDHL1, SR-786, and U937 cells were plated at a density of 5 × 104 cells/ml and cultured for 5 days prior to MTS assay. The SCARB1 blocking, LDLR blocking, and isotype control antibodies were added at dilutions of 1:1000 to 1:250. Ferrostatin-1 and deferoxamine (DFO) were obtained from Sigma Aldrich and added at a final concentration of 1 μM. MTS values were standardized to PBS control.
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