Intracellular GSH levels were measured using luminescence-based GSH-Glo™ Glutathione Assay kit (Promega #V6911). In this assay, luciferin derivative is converted into luciferin in the presence of GSH in a reaction that is catalyzed by glutathione S-transferase. The amount of light generated in the firefly luciferase-coupled reaction is proportional to the amount of GSH present in the sample. The assay was performed according to the manufacturer’s instructions. Briefly, 1000 cells were plated in flat-bottom 96-well plates and the following day, treated with the drugs. After approximately 40 h, the media was removed and GSH-Glo™ reagent was added in each well, followed by 30 min incubation. 100 μL reconstituted luciferin detection reagent was added and incubated for another 15 min. The bioluminescence was measured with the plate reader. Imidazole ketone erastin IKE (Selleckchem #S8877) was used as the positive control for ferroptosis. The level of ferroptosis is inversely proportional to the levels of GSH.
Imidazole ketone erastin ike
Manufactured by Selleck Chemicals
Sourced in China, United States
Imidazole ketone erastin (IKE) is a chemical compound used in laboratory research settings. It serves as a core functional ingredient with specific properties and applications, but a detailed description of its intended use would require further information that is not available within the scope of this request.
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Pd184352, by Merck Group (1 mentions)
4 protocols using imidazole ketone erastin ike
1
Measuring Intracellular Glutathione Levels
2
Comprehensive Ferroptosis Modulation Protocol
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Blasticidin (AppliChem GmbH), RSL3 (Selleckchem), ML210 (Tocris), ML162 (Caymann), Erastin (Biomol), Sulfasalazine (SAS) (MedChemExpress), Imidazole Ketone Erastin (IKE) (Sellekchem), Ferrostatin-1 (Sigma Aldrich), Liproxstatin-1 (Biozol), Necrostatin-1s (Abcam), zVAD (ENZO), iFSP1 (Cayman Chemicals), Tert-butylhydroquinone (TBHQ) (Sellekchem), AMG510 (MedChemExpress), ARS1620 (Chemgood), PD184352 (Sigma Aldrich), MK2206 (Sellekchem), DRAQ7 (Biolegend), BODIPY C11 (Invitrogen), H2DCFDA (Invitrogen), Dharmafect I (Dharmacon), Puromycin (Sigma), Doxycycline hydrochloride (Alfa Aesar), 4-hydroxy-tamoxifen (4OHT) (Sigma), Polybrene (Merck), CaCl2 (Sigma Aldrich), HBS (Sigma Aldrich), propidium iodide (Sigma).
3
Immunoblotting and Immunoprecipitation of Cell Death Regulators
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The following antibodies were used at the indicated dilution for immunoblotting (IB) and immunoprecipitation (IP) analyses: VDAC3 (1:800 for IB, 3 µg for IP; Proteintech, Wuhan, China), FBXW7 (1:800 for IB; 3 µg for IP, Proteintech), Ubiquitin (1:800 for IB, Proteintech), and β-Actin (1:8,000 for IB, Proteintech). Horseradish peroxidase (HRP) labeled secondary antibody conjugates were purchased from Molecular Probes (Abbkine, Beijing, China). Erastin (APExBIO, Beijing, China), ferrostatin-1 (Fer-1), rapamycin (Rapa), MK-2206, chloroquine (CQ), 3-MA, bafilomycin A1 (BafA1), MG132, and imidazole ketone Erastin (IKE) were obtained from Selleck Chemicals (Houston, TX).
4
Ferroptosis induction in esophageal cancer
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The ESCC cell lines ECA9706 and KYSE150 were purchased from Shanghai Institute of Cell Biology (Shanghai, China) and were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were maintained in an atmosphere containing 5% CO2 at 37°C. Imidazole ketone erastin (IKE, #S8877), liproxstatin-1 (Lip-1, #S7699), KX2-391 (Tirbanibulin, #S2700) and celecoxib (#S1261) were obtained from Selleck Chemicals (United States). BODIPY-C11 (581/591) (#D3861) was obtained from Invitrogen (United States).
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