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.
Ml162
Manufactured by Cayman Chemical
Sourced in United States
ML162 is a chemical compound used in laboratory settings. It functions as a selective inhibitor for a specific molecular target. The core purpose of ML162 is to facilitate research and investigation in relevant scientific fields.
Automatically generated - may contain errors
Lab products found in correlation
Sulfasalazine, by Merck Group (4 mentions)
Ferrostatin 1, by Cayman Chemical (4 mentions)
Liproxstatin 1, by Cayman Chemical (3 mentions)
Ml210, by Cayman Chemical (3 mentions)
Methionine, by Thermo Fisher Scientific (2 mentions)
L cystine, by Merck Group (2 mentions)
L methionine, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Erastin2, by Cayman Chemical (2 mentions)
Deferoxamine mesylate salt, by Merck Group (2 mentions)
Ruxolitinib, by Cayman Chemical (2 mentions)
Mouse ifnγ 485 mi, by R&D Systems (2 mentions)
Gemcitabine, by Selleck Chemicals (2 mentions)
Erastin, by Cayman Chemical (2 mentions)
Liperfluo, by Dojindo Laboratories (2 mentions)
Anti ifnγ xmg1.2, by Thermo Fisher Scientific (2 mentions)
L buthionine sulfoximine, by Merck Group (2 mentions)
Siinfekl peptide ova 257 264, by Merck Group (2 mentions)
Jak inhibitor 1, by Cayman Chemical (2 mentions)
10 protocols using ml162
1
Reagent Procurement for Cell Metabolism
2
Ferroptosis Induction and Cystine Deprivation
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Ferroptosis was induced by drug treatment with erastin (Cayman #17754), sulfasalazine (Sigma S0883), ML-162 (Cayman #20455), or RSL3 (Cayman #19288) at the concentrations indicated in the figure for one day, or by cystine deprivation. Cystine deprived media were prepared as described36 –38 using the DMEM, high glucose, no glutamine, no methionine, no cystine media (ThermoFisher Scientific, 21013024) with 10% dialyzed Fetal Bovine Serum (Sigma, F0392), L-methionine (Sigma, M5308, final concentration 30 mg/L), L-glutamine (ThermoFisher, #25030–081, final concentration 4mM), and with added L-Cystine (Sigma, C6727) at 200 μM for regular full media or at the concentrations indicated in the figures.
3
Ferroptosis Pathway Modulation Assay
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TVB-3664 (FASNi) or the inactive isomer (TVB-2632) were from Sagimet Biosciences (San Mateo, CA). ML162 (#20455) or Ferrostatin-1 (#17729) were from Cayman. ARS-1620 (HY-U00418) and Liproxstatin-1 (HY-12726) were from MedChemExpress. 16:0–18:1 PC (hereafter PC #850457P), Sodium palmitate (#P9767) and N-Acetyl-L-cysteine (#A7250) were from Millipore-Sigma. CellROX™ Green Reagent (#C10444) was from Thermo Fischer Scientific.
4
Chemical Compounds for Cell Death Study
5
Ferroptosis-associated Compounds Evaluation
6
Ferroptosis Induction and Inhibition Assay
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All cell lines used in this study were purchased from the American Type Culture Collection and were free of mycoplasma contamination (tested by the vendor). No cell line used in this study has been found in the International Cell Line Authentication Committee database of commonly misidentified cell lines, based on short tandem-repeat profiling performed by the vendor. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (vol/vol) fetal bovine serum (FBS) and 1% (vol/vol) penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37 °C as described previously (38 (link), 39 (link)). All cell lines were seeded into a 12-well plate for cell death and lipid peroxidation measurements. Cells were treated with ferroptosis inducers, including RSL3 (Selleck Chemicals), ML210 (Cayman Chemical), ML162 (Cayman Chemical), and FIN56 (Cayman Chemical); ferroptosis inhibitors, including liproxstatin-1 (Cayman Chemical) and ferrostatin-1 (Sigma-Aldrich); deferoxamine (Sigma-Aldrich); glycerol (Sigma-Aldrich); sn-G3P (Cayman Chemical); the GPD2 inhibitor iGP-1 - Calbiochem (Sigma-Aldrich); and antioxidants, including 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO; Sigma-Aldrich), mitochondria-targeted TEMPO (MitoTEMPO; Sigma-Aldrich), MitoQ (Cayman Chemical), and MitoQH2 (Cayman Chemical).
7
Compound Acquisition for Cell Assays
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Erastin, doxorubicin, rhodamine 123, and FIN56 were purchased from Sigma-Aldrich (St. Louis, MO). Erastin2, imidazole ketone Erastin, RSL3, ML-162, FINO2, GPX4 inhibitor 26a, JKE-1674, and JKE-1716 were from Cayman Chemical (Ann Arbor, MI). Valspodar was obtained from MedChemExpress (Monmouth Junction, NJ). Romidepsin was from Selleck Chemicals (Houston, TX). Purpurin-18 was purchased from Frontier Scientific (Logan, UT). Piperazine Erastin was from TargetMol (Wellesly Hills, MA). SN-38 was obtained from LKT Laboratories (St. Paul, MN). RSL3 was purchased from Tocris (Minneapolis, MN). Fumitremorgin C (FTC, > 95% purity) was synthesized in-house by the Developmental Therapeutics Program at the National Institutes of Health (Bethesda, MD).
8
Ferroptosis Induction and Cystine Deprivation
9
Ferroptosis-associated Compounds Evaluation
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Erastin, RSL3, ML162, ML210, ferrostatin-1, liproxstatin-1, JAK inhibitor I and ruxolitinib were purchased from Cayman Chemical. Doxorubicin (Adriamycin) HCl and gemcitabine were purchased from Selleckchem. Deferoxamine mesylate salt, L-Glutathione reduced, sulfasalazine, L-Buthionine-sulfoximine, 2-Mercaptoethanol, and SIINFEKL peptide (OVA 257–264) were purchased from Sigma-Aldrich. Recombinant human IFNγ (285-IF) and mouse IFNγ (485-MI) were purchased from R&D. BODIPY 581/591 C11, anti-IFNγ (XMG1.2) and anti-TNFα (MP6-XT22) blocking antibodies were purchased from Thermo Fisher Scientific. Liperfluo was purchased from Dojindo Molecular Technologies. Cyst(e)inase was obtained from the laboratory of Everett Stone and George Georgiou (University of Texas at Austin, TX, USA).
10
Cytotoxicity Screening of Small Molecules
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MDA-MB-231-Par and HCC1806-Par cells were seeded at 1,000 cells per well in quadruplicate per condition in a white opaque 96-well plate (catalog no. 3917, Corning). Twenty-four hours later, cells were treated with serial dilutions between 2.05pM and 100uM of the following compounds: neratinib (catalog no. 18404, Cayman Chemical), CAY10618, 1S,3R-RSL-3 (catalog no. 19288, Cayman Chemical), AZD7762 (catalog no. 11491, Cayman Chemical), ceranib-2 (catalog no. 11092, Cayman Chemical), and ML162 (catalog no. 20455, Cayman Chemical), and DMSO control. Cells were treated for 72 hours with media replaced every 24 hours. Cell viability was measured with the CellTiter-Glo 2.0 Assay (catalog no. G9243, Promega Corporation) with 1,000 ms integration time.
All cells were cultured at 37 °C in a humidified incubator with 5% CO2. MDA-MB-231 (ATCC HTB-26) cells were grown in DMEM supplemented with 10% FCS, penicillin (100 U ml−1), streptomycin (100 μg ml−1) and amphotericin (1 μg ml−1). HCC1806 (ATCC CRL-2335) cells were grown in Roswell Park Memorial Institute-1640 medium supplemented with 10% FCS, L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 U ml−1), streptomycin (100 μg ml−1) and amphotericin (1 μg ml−1).
All cells were cultured at 37 °C in a humidified incubator with 5% CO2. MDA-MB-231 (ATCC HTB-26) cells were grown in DMEM supplemented with 10% FCS, penicillin (100 U ml−1), streptomycin (100 μg ml−1) and amphotericin (1 μg ml−1). HCC1806 (ATCC CRL-2335) cells were grown in Roswell Park Memorial Institute-1640 medium supplemented with 10% FCS, L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 U ml−1), streptomycin (100 μg ml−1) and amphotericin (1 μg ml−1).
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