Cardiomyocyte death was estimated using a Live/Dead Viability/Cytotoxicity Assay Kit (Invitrogen). Isolated adult cardiomyocytes were pre-treated with or without 10 μM ferrostatin-1 (Sigma Aldrich: SML0583) for 30 min before treatment with 10 or 20 μM erastin (Sigma Aldrich: E7781), 10 or 100 μM isoproterenol (Sigma Aldrich: I5627), or 2 or 5 μM RSL3 (Sigma Aldrich: SML2234). The cells were then stimulated with or without ferrostatin-1 in the medium for four hours. After stimulation, the cells were stained with 1 mM calcein-AM (Invitrogen: C1430) and 2 μM ethidium homodimer-1 (Invitrogen: E1169) in the medium at 37°C for 10 min. The cells were washed three times using the medium and observed under a microscope (BZ-X700, Keyence). ROS production was measured by applying several indicators. After treatment with erastin or isoproterenol with or without ferrostatin-1, cells were stained with 25 μM 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen: D399) or C11-BODIPY (Invitrogen: D3861) in medium at 37°C for 10 min. The cells were then washed with the medium. ROS production was quantified using a fluorescence microplate reader.
Erastin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Erastin is a small molecule that functions as a potent and selective inducer of ferroptosis, a form of regulated cell death. It acts by inhibiting the cystine/glutamate antiporter system xc-, thereby depleting cellular glutathione levels and increasing lipid peroxidation, leading to cell death.
Automatically generated - may contain errors
Lab products found in correlation
Sodium dodecyl sulfate (sds), by Merck Group (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
Bca protein quantification kit, by Merck Group (3 mentions)
β actin, by Proteintech (3 mentions)
Primary antibodies against acsl4, by Proteintech (3 mentions)
Sp 900 general immunohistochemical kit, by ZSGB-BIO (3 mentions)
Cell tissue protein extraction kits, by Tiangen Biotech (3 mentions)
Acryl bis premixed powder, by Merck Group (3 mentions)
Horseradish peroxidase hrp substrate luminescent solution, by Merck Group (3 mentions)
Ultrasonic cleaner, by Thermo Fisher Scientific (2 mentions)
Rodent tail vein catheter, by Braintree Scientific (2 mentions)
Erastin c30h31cln4o4, by Selleck Chemicals (2 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
C11 bodipy, by Thermo Fisher Scientific (1 mentions)
Ethidium homodimer 1, by Thermo Fisher Scientific (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Ferrostatin 1, by Thermo Fisher Scientific (1 mentions)
Ferrostatin 1, by Merck Group (1 mentions)
Erastin, by Merck Group (1 mentions)
Bz x700, by Keyence (1 mentions)
7 protocols using erastin
1
Quantifying Cardiomyocyte Ferroptosis and ROS
2
Regulation of Ferroptosis in Ovine Granulosa Cells
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GCs were harvested from small follicles (1–2 mm in diameter) and large follicles (≥ 3 mm in diameter) in control lambs. GCs were resuspended in DMEM/F12 culture medium containing 10% foetal bovine serum and 1% antibiotic antifungal agent. Then, GCs were cultured at 37 ℃ and 5% CO2 in culture plates containing 2 × 105 cells per well.
The oar-miR-134-3p mimics, inhibitor, and NC (normal control) were purchased from Gemma Medical Technology Co., Ltd. (Shanghai, China). GCs from large follicles were seeded in a 24-well plate and transfected with oar-miR-134-3p mimics (100 nmol/μl), oar-miR-134-3p inhibitor (100 nmol/μl), and oar-miR-134-3p NC (100 nmol/μl) using Lipofectamine 3000 (Invitrogen, Shanghai, China) for 48 h. Subsequently, GCs were treated with or without erastin (ferroptosis inducer; Beyotime, Shanghai, China) for 24 h.
The oar-miR-134-3p mimics, inhibitor, and NC (normal control) were purchased from Gemma Medical Technology Co., Ltd. (Shanghai, China). GCs from large follicles were seeded in a 24-well plate and transfected with oar-miR-134-3p mimics (100 nmol/μl), oar-miR-134-3p inhibitor (100 nmol/μl), and oar-miR-134-3p NC (100 nmol/μl) using Lipofectamine 3000 (Invitrogen, Shanghai, China) for 48 h. Subsequently, GCs were treated with or without erastin (ferroptosis inducer; Beyotime, Shanghai, China) for 24 h.
3
Ferroptosis Induction and Detection
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Primary antibodies against ACSL4 and β-actin were purchased from Proteintech (Hubei Province, China). Erastin, a compound that could induce ferroptosis in mammalian cells, was purchased from Invitrogen (California, USA). The SP-900 general immunohistochemical kit was purchased from ZSGB-BIO (Beijing, China). Cell/tissue protein extraction kits were purchased from Tiangen Biotech (Beijing, China). Other chemicals and reagents such as MTT, sodium dodecyl sulfate, Acryl&Bis premixed powder, BCA protein quantification kit, and horseradish peroxidase (HRP) substrate luminescent solution were purchased from Sigma (St. Louis, MO, USA), Vetec (St. Louis, MO, USA) and Biological Industries (Kibbutz Beit Haemek, Israel).
4
Ferroptosis Induction and Detection
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Primary antibodies against ACSL4 and β-actin were purchased from Proteintech (Hubei Province, China). Erastin, a compound that could induce ferroptosis in mammalian cells, was purchased from Invitrogen (California, USA). The SP-900 general immunohistochemical kit was purchased from ZSGB-BIO (Beijing, China). Cell/tissue protein extraction kits were purchased from Tiangen Biotech (Beijing, China). Other chemicals and reagents such as MTT, sodium dodecyl sulfate, Acryl&Bis premixed powder, BCA protein quantification kit, and horseradish peroxidase (HRP) substrate luminescent solution were purchased from Sigma (St. Louis, MO, USA), Vetec (St. Louis, MO, USA) and Biological Industries (Kibbutz Beit Haemek, Israel).
5
Ferroptosis Induction and Detection
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Primary antibodies against ACSL4 and β-actin were purchased from Proteintech (Hubei Province, China). Erastin, a compound that could induce ferroptosis in mammalian cells, was purchased from Invitrogen (California, USA). The SP-900 general immunohistochemical kit was purchased from ZSGB-BIO (Beijing, China). Cell/tissue protein extraction kits were purchased from Tiangen Biotech (Beijing, China). Other chemicals and reagents such as MTT, sodium dodecyl sulfate, Acryl&Bis premixed powder, BCA protein quantification kit, and horseradish peroxidase (HRP) substrate luminescent solution were purchased from Sigma (St. Louis, MO, USA), Vetec (St. Louis, MO, USA) and Biological Industries (Kibbutz Beit Haemek, Israel).
6
Anti-Tumor Effects of Erastin and KRIBB3 in SCID Mice
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Indicated HeLa cells were subcutaneously injected into the dorsal flanks right of the midline in SCID mice (weight ~20g). At day seven, mice were injected with Erastin (20 mg/kg/ i.v., twice daily every other day) with or without KRIBB3 (50 mg/kg/ i.p., once daily every other day) for two weeks. The Erastin (C30H31ClN4O4) was bought from Selleck Chemicals. Erastin was dissolved in vehicle (2% DMSO and 98% phosphate buffered saline) and prepared by Ultrasonic Cleaner (Fisher Scientific). A final volume of 300 ul Erastin was applied through the tail vein. The half-life and area under curve (0-t) of Erastin are 27 min and 173 µg.min/ml, respectively. The Rodent Tail Vein Catheter (Braintree Scientific, MTV#1) were used to perform injection. This system allows easier access for both timed injections and repeated bolus administration. Tumors were measured once a week. The volumes were calculated using the following formula: volume (mm3)=length×width2×π/6. This study was approved by the Third Affiliated Hospital of Guangzhou Medical University and the University of Pittsburgh Institutional Animal Care and Use Committees and performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines (http://www.aaalac.org ).
7
Anti-Tumor Effects of Erastin and KRIBB3 in SCID Mice
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