For our in vitro inhibition assay, 1 × 104 cells/100 μl/well of H1299 cells sowed to 96 well plates were pretreated with the pan-caspase inhibitor z-VAD-fmk (R&D Systems, Minneapolis, MN, USA) (10 and 25 μM) for 1 h. After z-VAD-fmk was removed, H1299 cells were infected at MOI = 1 for 1 h. The virus suspension was removed, and 100 μl of fresh medium containing z-VAD-fmk (10 and 25 μM) was added to each well. After 24 h, cell viability was determined by CellTiter-Glo Assay following the manufacturer's instructions. Analysis was performed with the EnSpire Multimode Plate Reader.
Z vad fmk
Manufactured by R&D Systems
Sourced in United States, Germany, United Kingdom
Z-VAD-FMK is a caspase inhibitor that irreversibly binds to the catalytic site of caspase enzymes. It is commonly used in research applications to investigate the role of caspases in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Z ietd fmk, by R&D Systems (29 mentions)
Z lehd fmk, by R&D Systems (23 mentions)
Z devd fmk, by R&D Systems (19 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (15 mentions)
Cycloheximide (chx), by Merck Group (14 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Necrostatin 1, by Merck Group (11 mentions)
Z vdvad fmk, by R&D Systems (8 mentions)
Neon transfection system, by Thermo Fisher Scientific (8 mentions)
Streptomycin, by Thermo Fisher Scientific (8 mentions)
Propidium iodide, by Merck Group (8 mentions)
Bx795, by InvivoGen (7 mentions)
Bx795, by R&D Systems (7 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (7 mentions)
N acetyl l cysteine, by Merck Group (7 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Z aevd fmk, by R&D Systems (6 mentions)
Anti bcl xl, by Cell Signaling Technology (6 mentions)
Ferrostatin 1, by Merck Group (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
228 protocols using z vad fmk
1
Caspase Inhibitor Protects Cells from Viral Infection
2
Pharmacological Modulation of Retinal Regeneration
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hsp70 promoters were activated by immersing fish in a 37 °C water bath for 1 h, which was repeated every 6 h for the duration of the experiment. For treating fish with various pharmacological reagents, stock solutions were dissolved in DMSO. Vegfr inhibition was achieved with pazopanib (SelleckChem, 10 mM stock) by incubating fish in system water containing a maximum of 1 μM pazopanib starting 1 d prior to retinal injury and replacing with fresh pazopanib containing fish water daily (control: 0.0001% DMSO); mTor signaling inhibition with rapamycin (Sigma, 1 mM stock) was achieved by daily IP injections of 15 μL of a 25 μM stock in PBS beginning a couple of hours before retina injury (control: PBS containing 2.5% DMSO); Csf1r was inhibited with PLX3397 (SelleckChem, 100 mM stock) by incubating fish in system water containing a maximum of 1 μM PLX3397 starting 10 d prior to retinal injury and replacing with fresh PLX3397 containing fish water daily (control: 0.00001% DMSO); and caspase activity was inhibited with ZVAD-fmk (R&D Systems, 20 mM stock) by incubating fish in system water containing a maximum of 300 nM ZVAD-fmk just before retinal injury and replacing with fresh ZVAD-fmk containing fish water daily (control: 0.000015% DMSO).
3
Ebselen Cytotoxicity and Apoptosis Assay
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Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), obtained from Sigma-Aldrich Co. (CAS: 60940-34-3), is a compound with the chemical formula C13H9NOSe and a molecular weight of 274.18. Its purity level is indicated as greater than or equal to 98% according to TLC analysis. Ebselen was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co.) for experimental use at 100 mM as a stock solution. Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA) and dissolved in DMSO to generate a 10 mM stock solution. Cells were pretreated with Z-VAD for 1 h prior to treatment with Ebselen. DMSO (0.1%) was used as a vehicle control. All stock solutions were wrapped in foil and kept at 4 °C or −20 °C.
4
Assessing Cellular Stress Response Pathways
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All cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine and penicillin plus streptomycin. Tm, Tg, DTT and BfeA were purchased from Sigma and Tocris. zVAD-FMK was from R and D Systems. Caspase-Glo 3/7, Caspase-Glo 9, and CellTiter-Glo assay kits were from Promega. Antibodies to the RNase domain of IRE1α, (anti-IRE1α CD), ATF4, GAPDH, BAX, cleaved Caspase 3, BiP, and CHOP were from Cell Signaling Technology. Antibodies to phospho-IRE1α, XBP1s and IRE1α LD were generated at Genentech. Doxycycline was from Clontech. Geneticin selective antibiotic was from GIBCO.
5
Leishmania infantum Infection of Neutrophils
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Neutrophils were infected in vitro with L. infantum promastigotes stationary-phase at a ratio of 1:2 (neutrophil:parasites). For assays of cell death, mouse and human neutrophils were pretreated for 30 min with zVAD-fmk (100 µM) (R&D Systems, Minneapolis, MN, USA) or zIETD-fmk (100 µM) (R&D Systems, Minneapolis, MN, USA) to block caspase activation before infection. In some experiments, Nec-1 (50 µM), GSK’872 (3 µM), or NSA (10 µM), necroptosis inhibitors (all from Merck Millipore’s Calbiochem®, Darmstadt, Germany) were used. DMSO (vehicle) 0.4% (Cayman Chemical; Ann Arbor, MI, USA) was used as control. After 18 h, mouse infected neutrophils, or after 3 h, human infected neutrophils, were centrifuged, supernatants containing noninternalized promastigotes were collected, and medium was replaced by 250 µl Schneider insect medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 20% inactive FBS, 2 mM l -glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. After that, infected neutrophils were cultured at 25°C for an additional 3 days and intracellular load of L. infantum was estimated by production of proliferating extracellular motile promastigotes in Schneider medium (43 (link)).
6
Culturing MDA-MB-231 Cells with BAD
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MDA-MB-231 cells were from ATCC. Cells were cultured in RPMI 1640 medium (Life Technologies) with 10% FBS as previously described5 (link). Ectopic BAD expression and stable cell line generation was as before7 (link). Cell lines were routinely tested for mycoplasma contamination and passaged a maximum of 25 times. Z-VAD-FMK was from R&D systems, and necrosulfonamide was from Calbiochem.
7
Inhibition of Cell Death Pathways
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The MLKL inhibitor necrosulfonamide (NSA) was obtained from Tocris Bioscience (QL, United Kingdom). To inhibit caspases, the general caspase inhibitor Z-VAD-fmk was obtained from R&D Systems (Minneapolis, MN). The lipid oxidase inhibitor Liproxstatin-1, used to inhibit Ferroptosis, was obtained from Sigma (St Louis, MO). All inhibitor concentrations listed in figure legends. Cytospins were stained with PROTOCOL® HEMA 3® Stain set from Fisher Scientific (Kalamazoo, MI) according to the manufacturer's instruction. Antibody and chemical details provided in Table S1 .
8
Reagents for Apoptosis Induction Assays
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Human recombinant interferon beta (IFNβ) was obtained from R&D System (NY, USA). The primary mouse monoclonal antibody against TRAIL, clone 2E5 was purchased from Enzo Life Science (Paris, France). The caspase inhibitors: Z-VAD-fmk, Z-IETD-fmk and Z-DEVD-fmk and primary mouse monoclonal antibody against PD-1, clone 913,429 were obtained from R&D System (Wiesbaden, Germany). The primary mouse monoclonal antibody against FAS Ligand, clone NOK-1, was purchased from Abcam (Cambridge, UK). Anti-FAS antibody, clone ZB4, was obtained from Millipore (California, USA), anti-human B7-H1/PD-L1 monoclonal antibody, clone 130,021, and anti-human B7-DC/PD-L2 antibody, clone 176,611, were obtained from R&D System (Minneapolis, USA). To assess TRAIL- and FAS Ligand-mediated cytotoxicity, we used freshly eluted GST-TRAIL fusion protein, produced as described before [24] and FAS Ligand obtained from Enzo Life Science (Paris, France).
9
Necroptosis and Apoptosis Detection
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For the detection of MLKL phosphorylation, necroptosis was induced by treatment with PBZ complex (25 μg/ml poly(I:C) (Tocris, #4287); 1 μM BV6 (APExBIO, B4653); 20 μM z-VAD-fmk(APExBIO, A1902)) or TBZ complex (1000 units/ml, recombinant human TNF-alfa (TNF) (R&D Systems, 210-TA-020); 1 μM BV6; 20 μM z-VAD-fmk) for 4 h. Whole-cell lysates were collected and used for western blotting. For the detection of caspase 3 cleavage, apoptosis was induced by combined PB (25 μg/ml poly(I:C) and 1 μM BV6) or TB (1000 units/ml TNF and 1 μM BV6) treatment for various times. Whole-cell lysates were collected and western blotting was performed.
10
Analyzing T Cell Death Pathways
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T cell death was analyzed by flow cytometric staining for Annexin V (BD Bioscience) and 7-AAD (BD Bioscience) in different conditions. For in vitro activation, cells from the spleen or LNs were activated either with anti-CD3/CD28 signaling or with PMA/ionomycin stimulation. For treatment with Z-VAD-FMK (R&D Systems), BHA (Sigma), or okadaic acid (sigma), cells from LNs were pretreated with indicated concentrations of Z-VAD-ZMK, BHA or okadaic acid for 30min, then stimulated with PMA(10ng)/Ionomycin (1000ng) for 6h. For assessment of cell death in vivo, cells in the spleen or tumors from WT and KO mice were directly stained for Annexin V and 7-AAD without any in vitro stimulation.
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