Necrostatin-1

RIP1 kinase assay:

Phosphorylation of RIP1 requires its kinase activity. Expression constructs of FLAGtagged wild-type (WT) or a kinase-inactive pointmutant of RIP1 (K45M) are are transfected into 293T cells and RIP1 kinase assay is performed as described in the Methods in the presence of [γ-³²P]ATP for 30 min at 30℃. Samples are subjected to SDS-PAGE and RIP1 band is visualized by autoradiography. Relative intensities of radioactive bands are quantified and are shown (ratio) in this and all other autoradiographs. In parallel to kinase reactions, a sample of beads is subjected to western blot analysis using anti-RIP1 antibody to ensure equal protein amounts in kinase reactions.

• Cell lines: Jurkat, BALB/c 3T3, SV40-transformed MEF, L929
• Concentrations: 0.01-100 μM
• Incubation Time: --
• Method:

  Cells are seeded in 96-well plates (white plates for luminescent assays; black plates for fluorescent assays; clear plates for MTT assay) at the density of 5,000-10,000 cells per well for adherent cells or 20,000-50,000 cells per well for suspension cells in 100 μl of the appropriate phenol red-free media. After incubation, we determined cell viability using one of the following methods. For the ATP assay, we used luminescence-based commercial kits and analyzed luminescence using a Wallac Victor II plate reader. For Sytox assay, we incubated cells with 1 μM Sytox Green reagent for 30 min at 37℃, and then performed fluorescent reading. Subsequently, we added 5 μl of 20% Triton X-100 solution into each well to produce maximal lysis and incubated cells for 1 h at 37℃, then performed the second reading. We calculated the ratio of values before and after Triton treatment and normalized it to the relevant controls not subjected to cytotoxic stimuli, as indicated in figure legends. For the MTT assay, we used the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit. For PI exclusion assays, we added 2 μg/ml PI into the medium and immediately analyzed samples using FACSCalibur. For PI-annexin V assay we used the ApoAlert Annexin V-EGFP Apoptosis Kit. For DioC6 staining, we incubated cells with 40 nM DiOC₆ for 30 min at 37 ℃, washed once and analyzed in FACSCalibur. For ROS analysis, we incubated cells with 5 μM dihydroethidium for 30 min at 37 ℃, washed once and analyzed in FACSCalibur. EM analyses are performed at the Harvard Medical School EM facility. We acquired bright-field images of the cells using an Axiovert 200 microscope.

  
  (Only for Reference)