Necrostatin 2 is a potent necroptosis inhibitor. EC 50 for inhibition of necroptosis in FADD-deficient Jurkat T cells treated with TNF-α is 0.05 μM.Necrostatin 2 is also a RIPK1 inhibitor.
In Vitro
Evaluation of necroptosis inhibitory activity is performed using a FADD-deficient variant of human Jurkat T cells treated with TNF-α. Utilizing these conditions the cells efficiently undergo necroptosis, which is completely and selectively inhibited by Necrostatin 2 (EC 50 =50 nM). Necrostatin 2 shows activity in a broad range of necroptosis cellular systems. Necrostatin 2 at 30 μM completely protects L929 cells from TNF-α-induced necroptosis. In addition to TNF-α, the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD.fmk) has also been found to induce necrosis in L929 cells, which is efficiently inhibited by Necrostatin 2. EC 50 for inhibition of necroptosis in FADD-deficient Jurkat T cells treated with TNF-α is 0.05 μM. MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Cell Assay
L929 cells (100000 cells/mL, 100 μL/well in a 96-well plate) are treated with 10 ng/mL human TNF-α or 100 μM zVAD.fmk in the presence of DMSO (control), 30 μM Necrostatin 2, or 8for 24 h at 37°C in a humidified incubator with 5% CO 2 followed by ATP-based viability assessment as described in the previous experiment. Stock solutions (30 mM) in DMSO are initially prepared and then diluted with DMSO to give testing solutions. Each sample is done in duplicate. The final DMSO concentration is 0.5%. Cell viability values are adjusted to account for nonspecific toxicity, which in most cases is <10%. The reported cell viability values (%)±SD are determined from two independent experiments. MCE has not independently confirmed the accuracy of these methods. They are for reference only.
