The model system was used as previously described, in which 1.0 MPa compression was loaded on NP cells to imitate in vivo condition [15 (link), 16 (link)]. The cells were treated with DMSO (Control, Sigma, USA), necroptosis inhibitor Necronstatin-1 (Nec-1, Sigma, USA), autophagy inhibitor 3-Methyladenine (3-MA, Sigma, USA), and apoptosis inhibitor Z-VAD-FMK (Z-VAD, Merck, Germany) and then using a combination of the inhibitors: Nec-1+3-MA and Nec-1+Z-VAD. The bottom of the pressure vessel was filled with distilled water to preserve sufficient humidity and keep the device in an incubator at 37°C. 0 h mentioned in the experiment means the beginning of compression. The 0, 24, and 36 h compression-treated time points were selected in the current experiment according to our previous studies [15 (link), 16 (link)].
Z vad
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe, Morocco
Z-VAD is a laboratory reagent used for research purposes. It is a broad-spectrum caspase inhibitor that can be used to block cell apoptosis or programmed cell death. The core function of Z-VAD is to inhibit the enzymatic activity of caspases, which are crucial mediators of the apoptotic pathway.
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Lab products found in correlation
Cycloheximide (chx), by Merck Group (7 mentions)
Nec 1, by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Necrostatin 1, by Merck Group (4 mentions)
Chloroquine, by Merck Group (3 mentions)
N acetylcysteine, by Merck Group (3 mentions)
N acetyl cysteine (nac), by Merck Group (2 mentions)
Mg132, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Curcumin, by Merck Group (2 mentions)
N acetyl l cysteine, by Merck Group (2 mentions)
Staurosporine, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Doxorubicin, by Merck Group (2 mentions)
Fisetin, by Merck Group (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti zbp1, by R&D Systems (2 mentions)
Anti mlkl, by BD (2 mentions)
Anti hmgb1, by Thermo Fisher Scientific (2 mentions)
Deferoxamine, by Merck Group (2 mentions)
36 protocols using z vad
1
Mechanobiology of Nucleus Pulposus Cells
2
Glioblastoma Endothelial Cell Modulation
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Glioblastoma-induced endothelial cells were treated with EMAP II (Sigma–Aldrich, St. Louis, MO, United States) at concentrations of 0.005, 0.05, 0.5, and 5 nM (dissolved in 0.9% sodium chloride) for 0.5, 1, 3, 6, 12 h, and then the medium was replaced with fresh medium for 24 h. The cells of control group were treated with 0.9% sodium chloride. According to the results in our present study, 0.05 nM and 3 h was the optimal concentration and time, respectively. In addition, cells were pretreated with 3-MA (2 mM), Z-VAD (100 μM), 3-MA (2 mM) + Z-VAD (100 μM) and Baf-A1 (50 nM) (Sigma–Aldrich, St. Louis, MO, United States).
3
Neuroprotective Mechanisms against OGD Injury
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To test the effects of Nec-1 (20 μM, Sigma), BHA (100–200 μM, Sigma), NAC (0.05–10 mM, Sigma), and KN-93 (10 μM, Sigma), cells were pretreated with each of the reagents at the mentioned concentrations together with zVAD (20 μM, Sigma) for 1 h, followed by OGD insult. Control groups were treated with vehicle (DMSO, Sigma) together with zVAD for 1 h, followed by OGD insult. This dosing schedule of drugs was selected based on the results of previous reports.12 (link), 30 (link), 31 (link)To initiate OGD, cultures were switched to BDM medium that lacked glucose (Invitrogen) and were transferred to a chamber filled with 94% N2/5% CO2/1% O2 at 37 °C. Following OGD for 2.5 h, d -glucose was added back to the cultures to a final concentration of 25 mM, and the cultures were returned to an air/5% CO2 incubator at 37 °C. Cell death was assessed by PI staining, EM, and a cell membrane leakage assay. Molecular changes were examined by western blotting, immunofluorescent staining, and immunoprecipitation at the indicated time points after OGD insult.
4
Investigating Cell Death Pathways in HT-22 Cells
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Mouse hippocampal HT‐22 cells were cultured in DMEM (cat. no., 11995065, Gibco; Thermo Fisher Scientific, Inc) supplemented with 10% foetal bovine serum (cat. no., 10091148 Gibco; Thermo Fisher Scientific, Inc) and 1% antibiotic/antimycotic (cat. no., 15140122 Gibco; Thermo Fisher Scientific, Inc) at 37°C in 5% CO2. Recombinant TNF‐α was purified as described previously.8 z‐VAD was purchased from Merck Millipore, and propidium iodide was purchased from Sigma‐Aldrich (Merck KGaA). The following antibodies were used for western blotting: anti‐RIP1 (cat. no. 3493s; Cell Signaling Technologies, Inc), anti‐p‐MLKL (cat. no., ab196436; Abcam) and anti‐GAPDH (cat. no., AC033; AbClon, Inc).
5
Silibinin's Effects on PDGF-Induced Signaling
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Confluent cultured cells were preincubated with or without tunicamycin (T7765, Sigma-Aldrich, St. Louis, MO, USA), chloroquine diphosphate salt (CQ; Sigma-Aldrich, St. Louis, MO, USA), benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD; EMD Millipore, Billerica, MA, USA), or MG132 (Sigma-Aldrich, St. Louis, MO, USA) for 18 h. The cells were then incubated with or without 50 or 100 μM silibinin for 24 h before stimulation with 20 ng/mL PDGF (PeproTech, Rocky Hill, NJ, USA) for 24 h. Treated and untreated cells were washed with PBS, harvested by scraping, and centrifuged at 1000 g. Cell pellets were resuspended and sonicated in cold lysis buffer (PRO-PREPTM Protein Extraction Solution; iNtRON Biotechnology, Korea). The lysates were centrifuged at 12,000 g for 10 min, and the protein concentration in the clear supernatant was determined using BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA).
6
Bovine Macrophage Infection Assays
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The bovine macrophages were cultured on 24-well plates, at a density of 3 × 105 cells per well, to perform infection assays. We used multiplicities of infection (MOIs) of 2:1 and of 10:1 for the NY-1 ncp-BVDV strain and 2:1 for NADL cp-BVDV strain, with 500µL of RPMI medium supplemented with 10% heat-inactivated FBS (Gibco, New York, NY, USA). The negative controls were macrophages cultured with RPMI and 10% FBS, and the positive control was treated with 300 ng/mL of LPS from E. coli type O26:B26. For the inhibition treatments, the macrophages were pre-incubated for 2 h with CRID3 (Cytokine Release Inhibitory Drug 3, 50 µM; Sigma Aldrich, St. Louis, MO, USA), Y-VAD (50 µM; Merck, Boston, MA, USA) or Z-VAD (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone, 50 µM; Sigma Aldrich, St. Louis, MO, USA) and ODN A151 (5′-TTAGGGTTAGGGTTAGGGTTAGGG-3′) (3 µM, 6 µM and 12 µM). The supernatants were recovered at 24 h post-infection.
7
Curcumin Modulates Cellular Stress Responses
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Curcumin, AO, MDC, NAC, z-VAD, CHX, and DCF-DA were purchased from Sigma (St. Louis, MO, USA). The class III PI3K inhibitor 3-MA was obtained from Calbiochem (La Jolla, CA, USA). The CellTiter 96 AQueous One Solution Proliferation Assay System was purchased from Promega (Madison, WI, USA). Antibodies against PARP, caspase-3, p62, Beclin-1, CHOP, phospho-eIF2α (Ser51), IRE1-α, Bip/GRP78 and Nrf2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against LC3 (Sigma) were also used. The anti-β-actin, anti-ubiquitin, goat anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
8
Antibody-Based Protein Detection Protocol
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Antibodies against β-Actin, Bcl2, p-ERK, ERK, LC3, p62, XIAP, ATG5 and PARP were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-XIAP and anti-pP27 were purchased from BD Biosciences (San Jose, CA, USA). Anti-Caspase-3 (Active) antibody was purchased from Millipore (Temecula, CA, USA). An anti-ARHI murine monoclonal antibody (ID8) was generated in our laboratory. Doxycycline hyclate (DOX), chloroquine diphosphate salt (CQ), cis-diammine-platinum (II) dichloride (Cisplatin), Z-VAD, Nec-1 and N-acetyl-L -cysteine were purchased from Sigma-Aldrich (St. Louis, MO, USA).
9
Characterization of p62 Knockout MEFs
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HeLa and HEK293E cells were obtained from ECACC (European Collection of Cell Cultures). p62 knockout (p62−/−) and wild-type (p62+/+) mouse embryonic fibroblasts (MEFs) were kindly provided by Eiji Warabi of the University of Tsukuba. HEK293FT lentivirus packaging cells were from Invitrogen (Paisley, UK). Cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma #D6546) supplemented with 10% heat-inactivated FCS (Foetal Calf Serum, Biosera), 5% penicillin/streptomycin (Invitrogen) and 2mM l -glutamine (Sigma) in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were treated with puromycin (Santa Cruz Biotechnology), staurosporine (Sigma), z-VAD (Sigma), H2O2 (Sigma; the H2O2 stock was replaced every 2 weeks for the duration of the project), PR-619 (LifeSensors), curcumin (Sigma), auranofin (Sigma), N-acetylcysteine (Sigma), bafilomycin A1 (Enzo Life Sciences), chloroquine (Sigma), cycloheximide (Sigma) and retinoic acid (as an inhibitor of Nrf247 (link)) at different concentration and time-points as indicated. Medium was switched to serum-free DMEM for the duration of the treatments.
10
Cytotoxicity Assay Protocol for U2OS and HCT116 Cells
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U2OS cells were cultured in DMEM (Gibco/Life Technologies) supplemented with 10% FCS and antibiotics. siRNA-mediated knockdown was performed by reverse transfection of U2OS and HCT116 cells with 10 nM Silencer Select siRNAs (all Ambion/Life Technologies) using Lipofectamine 2000 (Invitrogen/Life Technologies). Plasmid transfection of U2OS cells was performed using Lipofectamine 2000 (Invitrogen/Life Technologies). For chemotherapeutic treatment, we used cisplatin (Teva), gemcitabine (Actavis) and neocarzinostatin (Sigma). For caspase inhibition, we used Z-VAD (Sigma) and for proteasome inhibition MG132 (Calbiochem). Control cells were treated with the same amount of solvent.
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