tert-Butyl hydroperoxide, cumene hydroperoxide, hydrogen peroxide, ferrostatin-1, cyclosporine A, GSH, GSSG, and MG132 were from Sigma. Doxorubicin hydrochloride, liproxstatin-1, deferoxamine, mitoQ, and SKQ1 were from Cayman Chemical. MitoPeDPP and mito-FerroGreen were from Dojindo. Necrostatin-1s was from Cell Signaling Biotechnology. MitoSOX, propidium iodide, and Hoechst 33,342 were from Invitrogen. The following antibodies were used: anti-HMGB1 (3935), anti-HO-1 (82,206), anti-VDAC (4661), anti-catalase (14,097), and anti-GAPDH (2118) from Cell Signaling Biotechnology; anti-Bach1 (sc-271211) from Santa Cruz Biotechnology; Anti-FTMT (PAD251Mu01) from Cloud-Clone Corp.; anti-GPX4 from R&D Systems.
Necrostatin 1s
Manufactured by Cell Signaling Technology
Sourced in United States
Necrostatin-1s is a potent and selective inhibitor of necroptosis, a form of programmed cell death. It acts by inhibiting the activity of receptor-interacting protein kinase-1 (RIPK1), a key regulator of the necroptosis pathway. Necrostatin-1s is used as a research tool to study the mechanisms and roles of necroptosis in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Ferrostatin 1, by Merck Group (2 mentions)
Cyclosporine a, by Merck Group (1 mentions)
Cumene hydroperoxide, by Merck Group (1 mentions)
Doxorubicin hydrochloride, by Cayman Chemical (1 mentions)
Liproxstatin 1, by Cayman Chemical (1 mentions)
Mitopedpp, by Dojindo Laboratories (1 mentions)
Anti bach1 sc 271211, by Santa Cruz Biotechnology (1 mentions)
Anti gpx4, by R&D Systems (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Anti gapdh 2118, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
Tert butyl hydroperoxide, by Merck Group (1 mentions)
Deferoxamine, by Cayman Chemical (1 mentions)
Mito ferrogreen, by Dojindo Laboratories (1 mentions)
Mitoq, by Cayman Chemical (1 mentions)
Zombie aqua, by Thermo Fisher Scientific (1 mentions)
3 protocols using necrostatin 1s
1
Oxidative Stress and Cell Death Modulators
2
Assessing Cell Viability and Apoptosis
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For viability measurement, cells were plated at 100,000 cells/mL in medium containing DMSO or 0.5 mM OSU-13 and incubated for 24, 48, and 72 hours (h) at 37°C and 5% CO2. Afterwards, cells were stained with Zombie-aqua (Life Technologies; Carlsbad, CA, USA) according to manufacturer’s instructions. Cells were then analyzed using an Attune Nxt cytometer (Invitrogen; Waltham, MA, USA), and FlowJo software (FlowJo LLC; Ashland, OR, USA).
For apoptosis and necroptosis inhibition assays, OPM-2 cells (250,000 cells/mL) were pre-incubated for 1 h at 37oC in medium containing 100 mM of the general caspase inhibitor Z-VAD-FMK (Sigma-Aldrich; St Louis, MO, USA) or the necroptosis inhibitor necrostatin-1s (Cell Signaling Inc.; Danvers, MA, USA). Then, cells were washed and incubated for 72 h in medium containing DMSO or 1 mM OSU-13, stained with Zombie-aqua (Life Technologies), and cell viability was assessed as previously described.
For apoptosis and necroptosis inhibition assays, OPM-2 cells (250,000 cells/mL) were pre-incubated for 1 h at 37oC in medium containing 100 mM of the general caspase inhibitor Z-VAD-FMK (Sigma-Aldrich; St Louis, MO, USA) or the necroptosis inhibitor necrostatin-1s (Cell Signaling Inc.; Danvers, MA, USA). Then, cells were washed and incubated for 72 h in medium containing DMSO or 1 mM OSU-13, stained with Zombie-aqua (Life Technologies), and cell viability was assessed as previously described.
3
Synthesis and Characterization of TH-2-31
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RSL3, IKE were synthesized by the Stockwell lab23 (link),29 (link). All other chemicals were purchased from qualified vendors: staurosporine, Selleck Chemicals S1421; Z-VAD-FMK, Abcam ab120382; LCL-161, ChemieTek CT-LCL161; TNFα, PeproTech 300-01 A; ferric ammonium citrate, Sigma-Aldrich F5879; hemoglobin, Sigma-Aldrich H7379; ferrostatin-1, Sigma-Aldrich SML0583; Liproxstatin-1, Selleck Chemicals S7699; necrostatin-1s, Cell Signaling Technology 17802 S. TH-2-31 (N2,N3-dicyclohexyl-5-(3-methyl-1,2,4-oxadiazol-5-yl)pyridine-2,3-diamine) was synthesized by Curia. The structure of Th-2-31 was confirmed by NMR: 1H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 1.7 Hz, 1H), 6.86 (d, J = 1.9 Hz, 1H), 4.04–3.78 (m, 1H), 3.19 (td, J = 10.0, 4.2 Hz, 1H), 2.44 (s, 3H), 2.01 (d, J = 10.6 Hz, 4H), 1.89 – 1.64 (m, 4H), 1.58 (d, J = 13.0 Hz, 1H), 1.47 – 1.01 (m, 9H).
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