| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| 250mg |
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| 500mg |
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| Other Sizes |
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Purity: ≥98%
| Targets |
RIP1 kinase (EC50 = 182 nM)
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| ln Vitro |
Necrostatin-1 (1-100 μM) prevents endogenous and overexpressed RIP1 from being autophosphorylated.It has been discovered that Necrostatin-1's antinecroptosis activity is primarily mediated by RIP1. [1]
Necrostatin-1 effectively prevents necroptotic cell death in a range of cell types that is induced by a variety of stimuli. With an EC50 of 490 nM, necrostatin-1, previously identified as a small-molecule necroptosis inhibitor, blocks both RIP kinase and TNF-α-induced necroptosis in Jurkat cells. [2]
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| ln Vivo |
Necrostatin-1 (Nec-1) is a specific small molecule inhibitor of receptor-interacting protein kinase 1 (RIPK1), which prevents RIPK1's phosphorylation.
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| Enzyme Assay |
Phosphorylation of RIP1 requires its kinase activity. RIP1 kinase assays are carried out as outlined in the Methods in the presence of [γ-32P]ATP for 30 min at 30°C using expression constructs of FLAGtagged wild-type (WT) or a kinase-inactive pointmutant of RIP1 (K45M) or FLAGtagged wild-type (WT) or K45M. Following SDS-PAGE, samples are subjected to autoradiography to identify the RIP1 band. This autoradiograph, along with all others, displays the ratio-based relative intensities of radioactive bands. To make sure that there are equal amounts of protein in kinase reactions, a sample of beads is put through a western blot analysis using anti-RIP1 antibody.
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| Animal Protocol |
Cells are seeded at a density of 5,000–10,000 cells per well for adherent cells or 20,000–50,000 cells per well for suspension cells in 96-well plates (white plates for luminescent assays, black plates for fluorescent assays, clear plates for MTT assay) in 100 l of the appropriate phenol red-free media. We used one of the following techniques to assess cell viability following incubation. We used luminescence-based commercial kits for the ATP assay and a Wallac Victor II plate reader to analyze luminescence. For the Sytox assay, cells were incubated with 1 M Sytox Green reagent for 30 min at 37°C before fluorescent reading was carried out. We then added 5 μl of a 20% Triton X-100 solution to each well in order to achieve the greatest amount of lysis. Cells were then incubated for 1 h at 37°C before the second reading was taken. We determined the ratio of values before and after Triton treatment and normalized it to the pertinent controls not exposed to cytotoxic stimuli, as shown in the figure legends. CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit was utilized for the MTT assay. We immediately analyzed samples using the FACSCalibur after adding 2 g/ml PI to the medium for PI exclusion assays. The ApoAlert Annexin V-EGFP Apoptosis Kit was utilized for the PI-annexin V assay. We stained cells with 40 nM DiOC6 for 30 min at 37 °C, then washed them once and ran a FACSCalibur analysis. For ROS analysis, we incubated cells with 5 μM dihydroethidium for 30 min at 37 ℃, washed once and analyzed in FACSCalibur. The Harvard Medical School EM facility is where EM analyses are carried out. An Axiovert 200 microscope was used to capture bright-field images of the cells.
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| References |
| Molecular Formula |
C13H13N3OS
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|---|---|---|
| Molecular Weight |
259.33
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| Exact Mass |
259.07793
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| Elemental Analysis |
C, 60.21; H, 5.05; N, 16.20; O, 6.17; S, 12.36
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| CAS # |
4311-88-0
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| Related CAS # |
Necrostatin-1 (inactive control);64419-92-7
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| Appearance |
Light yellow solid powder
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| SMILES |
CN1C(=O)C(NC1=S)CC2=CNC3=CC=CC=C32
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| InChi Key |
TXUWMXQFNYDOEZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H13N3OS/c1-16-12(17)11(15-13(16)18)6-8-7-14-10-5-3-2-4-9(8)10/h2-5,7,11,14H,6H2,1H3,(H,15,18)
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| Chemical Name |
5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylideneimidazolidin-4-one
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (9.64 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: 5% DMSO+45% PEG 300+ddH2O: 10mg/mL Solubility in Formulation 4: 12.5 mg/mL (48.20 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: 1.67 mg/mL (6.44 mM) in 10% (50% EtOH 50% Cremophor EL) 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8561 mL | 19.2805 mL | 38.5609 mL | |
| 5 mM | 0.7712 mL | 3.8561 mL | 7.7122 mL | |
| 10 mM | 0.3856 mL | 1.9280 mL | 3.8561 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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