Human colonoids were grown in WNR CM and passaged as above. After 3 days in culture, media was changed to WNR CM containing DMSO (vehicle) or 10 ng/ml TNFα (R & D Systems, Minneapolis, MN). + 80 uM pan caspase inhibitor z-VAD-FMK (zVAD, R & D Systems) + 2 uM AZD 5582 second mitochondria-derived activator of caspase (Smac) mimetics (Tocris, Minneapolis, MN). The main endpoints of interest were measured 24 h later. Cell viability was determined with imaging using a BZ-X microscope (Keyence, Itasca, IL) and CellTiter-Glo 3D reagent (Promega, Madison, WI). Supernatants were collected for HMGB1 analysis by ELISA (IBL International/Tecan, Hamburg, Germany) and cytokine analysis by V-plex immunoassay (Meso Scale Diagnostics, Rockville, MD). For protein expression, cells were lysed with lysis buffer (Cell Signaling Technologies, Danvers, MA) plus HALT protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Protein concentration was determined with BCA kit (ThermoFisher Scientific). Phosphorylated MLKL (pMLKL) was determined by western blotting using an anti-pMLKL (Ser358) antibody (Cell Signaling Technology, 91689, clone D6H3V). Anti-b-actin antibody (Cell Signaling Technologies, clone 8H10D10) was used as a loading control.
Z vad fmk zvad
Manufactured by R&D Systems
Sourced in United States
Z-VAD-FMK is a broad-spectrum caspase inhibitor that irreversibly binds to the catalytic site of caspase enzymes, thereby inhibiting their activity. It is a commonly used tool compound in cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Nec 1, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Smac mimetics, by Bio-Techne (1 mentions)
V plex immunoassay, by Mesoscale (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Bz x microscope, by Keyence (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Celltiter glo 3d reagent, by Promega (1 mentions)
B16 blue ifnα β reporter cell line, by InvivoGen (1 mentions)
Verikine mouse ifn β elisa kit, by PBL Assay Science (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Tiron, by Merck Group (1 mentions)
Necrosulfonamide nsa, by Bio-Techne (1 mentions)
Recombinant human ifn γ, by R&D Systems (1 mentions)
E coli o111 b4 lps, by InvivoGen (1 mentions)
Necrostatin 1 nec 1, by Santa Cruz Biotechnology (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
8 protocols using z vad fmk zvad
1
Investigating Necroptosis in Colonoids
2
Murine Macrophage Type I IFN Response
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BMD-Mφ were stimulated with 100ng/mL of LPS (Sigma-Aldrich) for 24 hours or with 1μg/mL of 5’triphosphate (ppp) dsRNA (InvivoGen) for different lengths of time. Secretion of total active type I IFN (both IFN-α and IFN-β) in BAL fluid, lung homogenates and cell culture supernatants was assessed using B16-blue IFNα/β reporter cell line for murine samples or HEK-blue IFNα/β reporter for human samples (InvivoGen), according to the specifications of the manufacturer. IFN-β levels in culture supernatants were measured using Verikine Mouse IFN-β ELISA kit (PBL Assay Science #42400–1). When indicated, BMD-Mφ were pre-treated with a combination of necrostatin-1 inhibitor (Nec-1, 10μM, Sigma-Aldrich), zVAD-FMK (zVAD, 25μM, R&D) for 1 hour before infection with IAV. In some experiments, BMD-Mφ were pre-treated with the selective RIPK3 inhibitor GSK’843 (kindly provided by GSK) (10μM) [52 (link)]. Samples were then collected for further analysis.
3
Otic Delivery of Pan-Caspase Inhibitor and Necroptosis Blocker
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Stock solutions of the pan-caspase inhibitor Z-VAD-FMK (ZVAD) (R&D Systems, Minneapolis, MN, USA, FMK001) and Nec-1 (Sigma-Aldrich, St. Louis, MO, USA, n9037) were made in DMSO. Immediately before use, each stock solution was diluted in 0.9% saline. The final concentrations of ZVAD and Nec-1 were 100 μM and 300 μM, respectively, based on the highest concentration without hair cell damage in mouse explant cultures. These concentrations may not be the optimal concentration for the local delivery of ZVAD and Nec-1 to the middle ear of the adult mice. Surgical procedures as described above requiring ∼15 min were conducted immediately following noise exposure and the experimental solution was delivered to fill the otic bulla of the left ear fully. Control mice underwent the same surgical procedure with delivery of identical concentrations of vehicle solution, DMSO diluted in saline, also to the left ear. One hour after noise exposure, the animals were euthanized and the temporal bones were removed to dissect the cochlea for the immunofluorescence assay.
4
Inhibiting Inflammatory Pathways in Cell Culture
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A total of 10 ng/ml human recombinant-IFNγ (R&D Systems, Minneapolis, Minnesota, USA) was added to the cells 24 h before infection. Moreover, 50 μM z-VAD-fmk (zVAD) (R&D Systems), 50 μM z-YVAD-fmk (YVAD) (R&D Systems), 25 μM necrostatin-1 (Nec1) (Santa Cruz Biotechnology, Dallas, Texas, USA), 5 μM necrosulfonamide (NSA) (Tocris), 200 nM cytochalasin D (Sigma-Aldrich), 1 mM EGTA (Sigma-Aldrich), 0.5 mM ATP (Sigma-Aldrich), 5 μM MCC950 (Tocris), 200 μM Tiron (Sigma-Aldrich), 50 mM N,N′-dimethylthiourea (DMTU) (Sigma-Aldrich), 10 μM oligomycin (Sigma-Aldrich) or 2 μM staurosporine (STS) (Calbiochem, San Diego, California, USA) were added to the cells 30 min before infection. For co-treatment, ATP was added 30 min before infection, while EGTA or YVAD were added 45 min before infection. Ultrapure E. coli O111:B4 LPS (Invivogen, San Diego, California, USA) transfection was performed using Lipofectamine 2000 (Invitrogen, Waltham, Massachusetts, USA) at 5 μg/ml.
5
Apoptosis Induction Assay with Flow Cytometry
6
Chondrocyte Necroptosis Induction Protocol
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Cartilage explants from tissue of 27 patients (mean age 68 years, ranging 54–78 years) were subjected to a defined impact energy of 0.59 J by using a drop-tower model as previously described1 (link)–3 (link). In addition, we included a chemical induction by TNF-a (10 or 100 ng/mL; Gibco, Thermo Fisher Scientific, Schwerte, Germany) and simultaneous sensitization with translation inhibitor cycloheximide (CHX; 5 or 10 µg/mL; Sigma-Aldrich), as described in previous studies18 (link),19 (link). To distinguish between necroptotic and apoptotic processes, cartilage explants were continuously treated with 2 mM N-acetyl-L-cysteine (Sigma-Aldrich)1 (link) or 40 µM Nec-1 (Sigma-Aldrich) w/ and w/o and 20 µM zVAD-FMK (zVAD; R&D Systems, Wiesbaden, Germany) for 4 days. MLKL inhibitor Necrosulfonamide (NSA; Tocris Bioscience, Bristol, UK) was exemplarily tested at a concentration of 2.5 µM. Fresh additives were provided after 24 h. Optimum concentration of Nec-1 was evaluated in prior cell culture experiments (Supplementary Fig. S1 ).
7
AE-848 Mediated Apoptosis Signaling
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AE-848 was dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) at a concentration of 50 mM and stored at 37°C until use. Human CD38-PE monoclonal antibody was obtained from Miltenyi (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Annexin V-FITC/propidium iodide (PI) detection kit and mitochondrial membrane potential (MMP) detection kit were purchased from KeyGEN BioTECH (Jiangsu, China). MTT was obtained from Solarbio (Beijing, China). Antibodies against Caspase-8, Caspase-3, cleaved poly ADP-ribose polymerase (PARP), P65, phosphatidylinositol 3 kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), GAPDH and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). And antibodies against NF-κB2 P100/P52, NF-κB1 P105/P50, Rel-B, c-Rel and Histone-3 were purchased from Abcam (Cambridge, UK). Pan-caspase inhibitor, Z-VAD-FMK (zVAD), was purchased from R&D Systems (Minneapolis, MN, USA).
8
Investigating Cell Death Signaling Pathways
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