Z wehd fmk

Manufactured by R&D Systems

Sourced in United States

Z-WEHD-FMK is a synthetic peptide that functions as a cell-permeable and irreversible inhibitor of caspase-1, -3, -4, -5, -6, -7, -8, and -10. It binds to the active site of the caspases, preventing their catalytic activity.

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Z-WEHD-FMK (FMK002, (2 citations)

30 protocols using z wehd fmk

Caspase Inhibition Assay for DENV

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For inhibition of caspase-1 only, Z-WEHD-FMK Caspase-1 Inhibitor (R&D Systems) was diluted in sterile DMSO and cells were pre-treated with 80 μM Z-WEHD-FMK or DMSO vehicle 30 minutes prior to inoculation with DENV. Dilutions were made into cell culture medium.
For the caspase inhibition panel, Z-VAD-FMK (pan caspase inhibitor), Z-WEHD-FMK and Z-YVAD-FMK (caspases-1, -4, and -5 inhibitors), Z-DEVD-FMK (caspase-3 inhibitor), Z-IETD-FMK (caspase-8 inhibitor), and Z-LEHD-FMK (caspase-9 inhibitor) (all from R&D Systems) were diluted in sterile DMSO and given to the cells at a final concentration of 1 μM each at the time of inoculation with DIV crude supernatant.

Callaway J.B., Smith S.A., Widman D.G., McKinnon K.P., Scholle F., Sempowski G.D., Dittmer D.P., Crowe JE J.r., de Silva A.M, & Ting J.P. (2015). Source and Purity of Dengue-Viral Preparations Impact Requirement for Enhancing Antibody to Induce Elevated IL-1β Secretion: A Primary Human Monocyte Model. PLoS ONE, 10(8), e0136708.

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Neutrophil NETosis Induction by Platelet Supernatants

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According to previously reported methods (69 (link)), neutrophils were purified from mouse blood samples using Ficoll-Paque (Ficoll-Paque Plus, 1.077 g/mL, GE Healthcare, Chicago, IL, USA) and dextran (Sigma-Aldrich) sedimentation (3% w/v) density gradient centrifugation and red blood cell lysis. A flow cytometer (Gallios, Beckman Coulter, Brea, CA, USA) and a fluorescent anti-citrullinated histone H3 (CitH3) antibody (Abcam, Cambridge, UK) were used to investigate the neutrophil expression of NETosis marker CitH3 after treatments of supernatants from platelets or platelets plus NDs. To prepare the platelet supernatants, inhibitors (Z-WEHD-FMK, 10 μM, R&D Systems; Z-DEVD-FMK, 10 μM, R&D Systems; OLT1177, 10 μM, Cayman Chemical; NAC 150 ng/mL, Sigma-Aldrich; MitoTEMPO, 1 μM, Sigma-Aldrich; P-selectin: rP-sel, 100 ng/mL R&D Systems; 30 min pretreatments before addition of ND) were used to block ND-induced platelet activation and cell death. After treatments with or without NDs and inhibitors, platelet supernatants were harvested by centrifugation (2.5 x 10⁴ g, 10 min; Benchtop Centrifuge, ThermoFisher Scientific) to remove platelets and NDs. Peptidyl arginine deiminase 4 (PAD4) inhibitor GSK484 (10μM, Sigma–Aldrich, St. Louis, MO, USA) was used to block neutrophil NETosis in vitro and in vivo as described (69 (link)).

Hung S.C., Ke L.C., Lien T.S., Huang H.S., Sun D.S., Cheng C.L, & Chang H.H. (2022). Nanodiamond-Induced Thrombocytopenia in Mice Involve P-Selectin-Dependent Nlrp3 Inflammasome-Mediated Platelet Aggregation, Pyroptosis and Apoptosis. Frontiers in Immunology, 13, 806686.

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Leishmania Infection of Bone Marrow-Derived Macrophages

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Isolated femurs and tibia were flushed with PBS, and precursor cells were cultured in complete RPMI supplemented with 30% L929 cell-conditioned medium. After 7 d in culture, mature bone marrow-derived macrophages (BMDM) were harvested and infected with metacyclic promastigotes at different multiplicities of infection (MOI). For detection of IL-1β secretion by infected cells by ELISA, BMDMs were primed for 5 h with LPS (50 ng/ml), washed, and then infected with metacyclic promastigotes at different MOIs for 6 hr. Supernatants were assayed using the IL-1β DuoSet ELISA (R&D Systems). For western blot analysis of the active form of IL-1β, BMDMs were treated or not with LPS (50 ng/ml) and infected or not with metacyclic promastigotes at a 1:8 MOI for 6 hr. Supernatants were concentrated by methanol/chloroform precipitation and immunoblotted along with cell extracts for IL-1β (AF-401/R&D) and caspase-1 (M-20/Santa Cruz Biotech). For selective inhibition of caspase-1, BMDMs were pretreated with 20μM and 50 μM of Z-WEHD-FMK or control inhibitor Z-FA-FMK (R&D Systems) for 5 hr prior to infection.

Charmoy M., Hurrell B.P., Romano A., Lee S.H., Ribeiro-Gomes F., Riteau N., Mayer-Barber K., Tacchini-Cottier F, & Sacks D.L. (2016). The Nlrp3 inflammasome, IL-1β, and neutrophil recruitment are required for susceptibility to a non-healing strain of Leishmania major in C57BL/6 mice. European journal of immunology, 46(4), 897-911.

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Chorionic Villi Explant Culture

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Villous tissue was obtained from term uncomplicated pregnancies after caesarean section without labor (n=9) within 30 min after delivery and explants were prepared as previously described32 (link) with slight modifications. Briefly, biopsies of the chorionic villi were dissected and washed with PBS to remove any blood. Tissue was cut into small pieces (approx. 2-3mm) and three pieces (i.e. explants) were placed into netwells (74 μm mesh; Corning, Sigma-Aldrich, ON, Canada) in 1.5ml of culture media (RPMI, 5% heat-inactivated FBS, 100μg/ml streptomycin, 100IU/ml penicillin, 1μg/ml insulin, 0.1μg/ml hydrocortisone, 0.1μg/ml retinol acetate, 0.05mg/ml gentamycin; all chemical are from Sigma-Aldrich, ON, Canada). Explants were cultured at 37°C with 5% CO₂ with daily media change. On day 4, explants were treated with MSU crystals (100μg/ml; Invivogen, CA, USA), rhIL-1β (10ng/ml; Peprotech, Qc, Canada), caspase-1 inhibitor (10μM; Z-WEHD-FMK, R&D Systems, USA) or IL-1Ra (1μg/ml; Sobi-Swedish Orphan Biovitrum, ON, Canada) for 24 or 48h in Opti-MEM (Life Technologies, ON, Canada). Explants were processed for histology (fixed for 24h in 4% formalin and paraffin embedded) or protein analysis (supernatants and explants frozen and the stored at -80°C until extraction and analysis).

Brien M.E., Duval C., Palacios J., Boufaied I., Hudon-Thibeault A.A., Nadeau-Vallée M., Vaillancourt C., Sibley C.P., Abrahams V.M., Jones R.L, & Girard S. (2016). Uric acid crystals induces placental inflammation and alters trophoblast function via an IL-1-dependent pathway: implication for FGR. Journal of immunology (Baltimore, Md. : 1950), 198(1), 443-451.

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Macrophage Differentiation and Homocysteine Response

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THP-1 cells were cultured in RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone), as well as 100 U/ml penicillin and streptomycin. THP-1 cells were treated with phorbo 12-myristatel 13-acetate (80 nmol/l) to induce the differentiation of monocytes into macrophages. Cells were subsequently washed and incubated with different concentration of Hcy (Sigma, USA) for 24 h. In some experiments, macrophages were treated with Hcy after transfected with NLRP3 siRNA or scrambled siRNA for 4 h; or macrophages were treated with Hcy in the presence or absence of caspase-1 inhibitor Z-WEHD-FMK (20 μmol/l, RD, USA), or antioxidant NAC (50 mmol/l, Sigma).

Wang R., Wang Y., Mu N., Lou X., Li W., Chen Y., Fan D, & Tan H. (2017). Activation of NLRP3 inflammasomes contributes to hyperhomocysteinemia-aggravated inflammation and atherosclerosis in apoE-deficient mice. Laboratory Investigation; a Journal of Technical Methods and Pathology, 97(8), 922-934.

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Transfection and Inhibition of HIV-1 LTR

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We seeded HMG or HFMG at a density of 2.5×10⁵ cells /well in a 48 well plate. At day 6, cells were transfected with GU-rich RNA within the HIV LTR (ssRNA40)/lyovec (Invivogen Cat# tlrl-lrna40) and ssRNA41/lyovec (identical to ssRNA40 with the exception that uridines are replaced with adenosines) (Invivogen Cat# tlrl-lrna41) for 24h and 48h. Both cell supernatants for ELISA and cell lysates for western blot analysis were collected. For inhibitor treatments, caspase-1 inhibitor, Ac-YVAD-cmk (Invivogen Cat# inh-yvad) or Z-WEHD-FMK (R&D Systems Cat# FMK002) or MCC950 (Adipogen Cat# AG-CR1–3615), cells were pre-incubated for 2h at 37°C prior to addition of ssRNA40. In parallel, untreated and ssRNA41 treated cells were also incubated at 37°C.

Rawat P., Diedrich C.T, & Spector S.A. (2018). Human Immunodeficiency Virus Type-1 single-stranded RNA activates the NLRP3 inflammasome and impairs autophagic clearance of damaged mitochondria in human microglia. Glia, 67(5), 802-824.

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Glucose and Metformin Stock Preparation

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Sterile D-glucose and L-glucose in 45% solution was purchased from Sigma-Aldrich (St. Louis, MO, USA) and stored at room temperature. Metformin powder was purchased from Enzo Life Sciences (Farmingdale, NY, USA), reconstituted in distilled deionized water, re-filtered with 0.22μm filters, and stored at −20°C. The caspase −1 inhibitor (Z-WEHD-FMK) was purchased from R&D Systems (Minneapolis, MN).

Han C.S., Herrin M.A., Pitruzzello M.C., Mulla M.J., Werner E.F., Pettker C.M., Flannery C.A, & Abrahams V.M. (2014). Glucose and metformin modulate human first trimester trophoblast function:A model and potential therapy for diabetes-associated uteroplacental insufficiency. American journal of reproductive immunology (New York, N.Y. : 1989), 73(4), 362-371.

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Caspase Inhibition in PBMC Response

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PBMC from 16 TB-IRIS and 15 non-IRIS patients (week 2, non-microarrayed) were thawed, rested and counted as above. Lyophilized Caspase-1/4/5 irreversible peptide inhibitor (Z-WEHD-FMK; R&D systems, Minneapolis, MN) was dissolved in DMSO at 1mM stock concentration. PBMC were treated with the Z-WEHD-FMK peptide inhibitor (50μM) or DMSO vehicle control (50μM) for 1 hour at 37°C. Supernatant was then removed and replenished with a fresh stock of RPMI supplemented with 10% FCS. Samples were either stimulated or unstimulated with heat-inactivated MTB (H37Rv; MOI 1:1) overnight at 37°C. Tissue culture (TC) supernatant was harvested and purified by centrifugations twice at 2,000rpm. The residual PBMC pellets were lysed in lysis buffer on ice for 1h and recentrifuged to remove all cell debris. Cytokine concentrations in supernatants were quantified by Luminex assay. Concentrations of Caspase-1 (R&D systems) and IL-1α (eBioscience) in supernatant and Caspase-3 (eBioscience) and Caspase-5 (BioVision, Milpitas, CA) in PBMC lysate were measured by ELISA. The activity of Caspase-5 was expressed as the fold change in absorbance (OD405nm) in MTB-stimulated over unstimulated PBMC lysate as recombinant protein standards were not supplied in the assay kit.

Lai R.P., Meintjes G., Wilkinson K.A., Graham C.M., Marais S., Van der Plas H., Deffur A., Schutz C., Bloom C., Munagala I., Anguiano E., Goliath R., Maartens G., Banchereau J., Chaussabel D., O'Garra A, & Wilkinson R.J. (2015). HIV–tuberculosis-associated immune reconstitution inflammatory syndrome is characterized by Toll-like receptor and inflammasome signalling. Nature Communications, 6, 8451.

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MTT Assay for BEAS-2B Cell Viability

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Cell viability of BEAS-2B cells was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay as described previously [32 (link)]. Briefly, cells were seeded at a density of 2500 cells per well of a 96-well flat-bottomed cell culture plate and maintained at 37°C, 5% CO₂ overnight. The inhibitors SB203580 (10 μM; EMD Millipore), E-64-D (10 μM; Enzo Life Sciences, Lausen Switzerland), Pepstatin (5 μM; Roche Diagnostics, Mannheim, Germany), Dorsomorphin (1 μM; Sigma-Aldrich, Munich, Germany) or Z-WEHD-FMK (10 μM; R&D Systems, Wiesbaden-Nordenstadt, Germany) were added 1 h prior to re-exposition of BEAS-2B cells with 100 μg/ml of PM_2.5. After 72 h, 0.5 mg/ml MTT was added for 2 h before formazan crystals were dissolved in 100% DMSO. Absorbance at 595 nm was determined with a BIO-RAD iMark^™ Microplate Reader (BIO-RAD, Hercules, CA). Cell viability was displayed as the percentage of untreated control cells.

Dornhof R., Maschowski C., Osipova A., Gieré R., Seidl M., Merfort I, & Humar M. (2017). Stress fibers, autophagy and necrosis by persistent exposure to PM2.5 from biomass combustion. PLoS ONE, 12(7), e0180291.

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Pharmacological Modulation of Cell Death

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Necrostatin-1, -5, -7, Glycine, Coenzyme Q₁₀, Resveratrol, DPPC, DPI, NAC, rotenone, antimycin, and TTFA were obtained from Sigma (Aldrich, St. Louis, MO). Necrostatin-1s and GSK’872 were obtained from BioVision (Milpitas, California). Necrosulfonamide was obtained from Tocris Bioscience (QL, United Kingdom). Caspase inhibitors: Z-VAD-FMK, general caspase inhibitor; Z-WEHD-FMK, caspase-1 inhibitor; Z-VDVAD-FMK, caspase-2 inhibitor; Z-DEVD-FMK, caspase-3 inhibitor; Z-YVAD-FMK, caspase-4 inhibitor; Z-VEID-FMK, caspase-6 inhibitor; Z-IETD-FMK, caspase-8 inhibitor; Z-LEHD-FMK, caspase-9 inhibitor; Z-AEVD-FMK and caspase-10 inhibitor were obtained from R&D Systems (Minneapolis, MN). GW806742X, MLKL inhibitor was obtained from SYNkinase (Australia). All chemical were used at the stated concentration in the figure legends.

González-Juarbe N., Gilley R.P., Hinojosa C.A., Bradley K.M., Kamei A., Gao G., Dube P.H., Bergman M.A, & Orihuela C.J. (2015). Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia. PLoS Pathogens, 11(12), e1005337.

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