Necrostatin

Activation of apoptosis through the detection of cleaved caspases was performed on the FACSCaliber flow cytometer according to the protocol previously described [15] (link). Antibodies towards cleaved caspase-3 (Cell Signaling Technologies) and cleaved caspase-8 (Cell Signaling Technologies) were used to detect apoptotic induction. Necrostatin (Enzo Life Sciences) and zVAD (Millipore) were used at final concentrations of 10 μM and 20 μM, respectively, in cell culture medium prior to PI exclusion assays. Treatment of Necrostatin or zVAD was initiated 24 hours after siRNA transfection, and continued every 24 hours until the endpoint of the assay. PI exclusion assays were performed by collecting cells, washing in PBS, and incubating in samples in 2 μg/ml PI for 5 minutes before analyzing for PI positivity as a marker for cell death. At least 10,000 events were captured per experimental condition. Data were analyzed using CellQuest software (BD Biosciences) and error bars plotted represent standard deviation of replicate conditions.

Cells in 96 well plates were treated with TNFα (0-25ng/ml; Miltentyi Biotec), zVAD-fmk (30 µM, Promega), necrostatin (30 µM, Enzo) or Necrox-2 (30 µM Enzo) as indicated. For mycobacterial infections, cells were infected with GC1237 for the times indicated in figure legends, and the bacteria removed by gently washing once with warm PBS-1% FCS before replacing with complete medium containing the treatments as indicated. Plates were then washed 2x with PBS, fixed for 24 hours with 4% paraformaldehyde, washed with PBS and stained with crystal violet as previously described.¹⁹ (link)

The following reagents were used: Ivermectin (Sigma I8898-250MG), KN-93 (Tocris, 5215-1 MG), Cyclosporin A (Tocris, 1101–100 MG), KN-62 (Tocris, 1277-1 MG), PSB 069 (Santa Cruz, sc-204216 10 MG), DCPIB (Tocris, 1540–10 MG), A438079 (Tocris, 2972–10 MG), z-vad-fmk (Enzo Life Sciences, ALX-260-020-M001-1 MG), vx765 (InvivoGen, inh-vx765-1-10MG), oxATP (Sigma Aldrich, A6779–25MG), PPAD (Sigma Aldrich, P178–10MG), DPI (Sigma Aldrich, D2926–10 MG), NAC (Sigma Aldrich, A9165–25 MG), Suramin (Sigma Aldrich, S2671–100 MG), Necrostatin (Enzo Life Sciences, BML-AP309–0020–20 MG), Digoxin (Sigma Aldrich, D6003–100 MG), Apyrase (New England Biolabs, M0393S–10,000 milliunits), ATP (Sigma Aldrich, A2383–1G), Pannexin-1-Inhibitor (AnaSpec, 61911–1 MG), Probenecid (Sigma Aldrich, P8761–25 G), YOPRO-1 Iodide (Life Technologies, Y3603-1 mL), GSH (Sigma Aldrich, G4251–1 G).