HEPES, water (W3500), Dulbecco′s Modified Eagle Medium (DMEM), 4,6,-O-ethylidene-D-glucose (ETDG), anti-GFAP polyclonal antibody, cytosine arabinoside (Ara-C), LaCl3 (La3+), cytochalasin B (Cyto-B) and ethidium (Etd) bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA), whereas penicillin, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), streptomycin, Propidium (Prd) iodide, diamidino-2-phenylindole (DAPI), YO-PRO-1, goat anti-mouse Alexa Fluor 488/555 and goat anti-rabbit Alexa Fluor 488/555 were from Thermo Fisher Scientific (Waltham, MA, USA). IL-1β and TNF-α were obtained from Roche Diagnostics (Indianapolis, MI, USA). Normal goat serum (NGS) was purchased from Zymed (San Francisco, CA, USA). Anti-Cx43 monoclonal antibody (610061) was obtained from BD Biosciences (Franklin Lakes, NJ, USA). The mimetic peptides gap19 (KQIEIKKFK, intracellular loop domain of Cx43), gap19I130A (KQAEIKKFK, negative control), Tat-L2 (YGRKKRRQRRR-DGANVDMHLKQIEIKKFKYGIEEHGK, intracellular loop domain of Cx43), and Tat-L2H126K/I130N (YGRKKRRQRRR-DGANVDMKLKQNEIKKFKYGIEEHGK, negative control) were obtained from Genscript (Piscataway, NJ, USA).
Tnf α
Manufactured by Roche
Sourced in United States, Germany
TNF-α is a laboratory equipment product that measures the concentration of tumor necrosis factor alpha, a cytokine involved in the inflammatory response. It provides quantitative results to support research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Roche (2 mentions)
Tnf α, by Merck Group (2 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Interleukin 6 (il 6), by Merck Group (2 mentions)
Spectramax m5 microplate reader, by Molecular Devices (2 mentions)
Gap19, by GenScript (1 mentions)
Yo pro 1, by Thermo Fisher Scientific (1 mentions)
Anti gfap polyclonal antibody, by Merck Group (1 mentions)
Ethidium etd bromide, by Merck Group (1 mentions)
Diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Tat l2, by GenScript (1 mentions)
Normal goat serum (ngs), by Zymo Research (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Pyrrolidine dithiocarbamate pdtc, by Merck Group (1 mentions)
Tgf β1, by Roche (1 mentions)
Sb202190, by Merck Group (1 mentions)
Sp600125, by Merck Group (1 mentions)
U0126, by Merck Group (1 mentions)
Interleukin 6 (il 6), by Roche (1 mentions)
29 protocols using tnf α
1
Evaluating Connexin Mimetic Peptides in Cell Cultures
2
Cytokine-Mediated Signaling Pathway Regulation
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The cytokines TGF-β1, TNF-α and IL-6 (Roche Diagnostics; Indianapolis, IN, USA) were added at 0.1, 1, or 10 ng/ml to cancer cells in serum-free DMEM and incubated at 37°C in an atmosphere of 5% CO2 for 48 h or as indicated. Smad3 inhibitor (SIS3; Merck KGaA, Billerica, MA, USA; 1, 5 or 10 µM), 100 µM pyrrolidine dithiocarbamate (PDTC; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 1 µM wortmannin (Sigma-Aldrich; Merck KGaA), 10 µM SP600125 (Sigma-Aldrich; Merck KGaA), 10 µM U0126 (Sigma-Aldrich; Merck KGaA) or 10 µM SB202190 (Merck KGaA) were incubated with confluent HeLa cells for 1 h at 37°C in a 5% CO2 atmosphere. A total of 10 ng/ml TGF-β1 was then added and the cells were incubated for an additional 48 h.
3
Culturing and Treating Human Cortical Neurons
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We purchased HCN-1A, primary human cortical neuronal cells, from American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s modified Eagle’s medium (DMEM, ATCC) supplemented with 20 % bovine calf serum, 1 % antibiotic–antimycotic, and 1 % l -glutamine (Life Technologies, Grand Island, NY) was used to culture HCN-1A cells according to the ATCC recommendations. The HCN-1A cells were used in immunohistochemistry. During the course of our studies, ATCC discontinued the distribution of HCN-1A cells due to quick senescence of the cells and insufficient inventory. The HCN-1A cells were thus unavailable for use in other assays. HCN-1A cells were treated overnight (17 h) with either the 40 amino acid Aβ peptide (Aβ1−40, Bachem, Torrance, CA, stock solution in ultrapure water) at a concentration of 125 µg/ml or human recombinant tumor necrosis factor-alpha (TNF-α, Roche, Indianapolis, IN) at a concentration of 25 ng/ml. Control treatments included HCN-1A incubation with either inactive (reverse peptide order) Aβ peptide (Aβ40−1) (Bachem, stock solution in 10 % acetic acid) or vehicle alone (basal medium or basal medium containing an equivalent volume of 10 % acetic acid). Aβ was diluted in basal medium and incubated for 2 h at room temperature before administration, to allow the formation of toxic oligomers and fibrillar aggregates that are found in AD.
4
Selective Liver Irradiation Mouse Model
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All mice used in this study were male and 8‐12 weeks old with a body weight of 20‐28 g. Mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Wild‐type (wt) (C57BL/6J) mice received single‐dose selective liver irradiation (25 Gy, dose rate 2.4 Gy/min) with or w/o injection of TNF‐α (2 μg/mouse) (Roche, Penzberg, Germany). A single injection of TNF‐α was administered intraperitoneally (i.p.) 20 minutes before irradiation, as described previously.27 PECAM‐1‐knock‐out (ko) (B6.129S‐Pecam1Gt(OST16303)Lex/J) mice also received selective liver irradiation with a single dose of 25 Gy as described before.4 The animals were killed at regular intervals at 1‐48 hours after irradiation. Sham‐irradiated and PBS‐injected (i.p.) control animals (n = 3 per each time‐point) were studied simultaneously in all experiments.
All animal studies were reviewed and approved by the committee of the Central Institute for Animal Experiments of the University Medical Centre Goettingen, and the Lower Saxony State Office for Consumer Protection and Food Safety (No. 33.12‐42502‐04‐10/0158).
All animal studies were reviewed and approved by the committee of the Central Institute for Animal Experiments of the University Medical Centre Goettingen, and the Lower Saxony State Office for Consumer Protection and Food Safety (No. 33.12‐42502‐04‐10/0158).
5
Neonatal Rat Ventricular CMs: TNF-α Exposure
6
Cytokine-induced Inflammation in ARPE-19 Cells
7
NF-κB Activation Dynamics in HEK293 Cells
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HEK293 cells were plated in 24-well plates (Cell-Bind from Corning) and transfected with 50, 100, or 150 ng of pcDNA3.1 – PLpro plasmid, 50 ng pNFkB-luc, and 25 ng pRL-TK (Renilla control) per well in triplicate (Mirus LT-1). Cells were incubated for 12 hours, media removed, and fresh media containing TNFα (Roche) at a final concentration of 10 ng/ml was added and incubated with cells for 4 hours. Cells were then lysed with passive lysis buffer and wells assayed for dual luciferase expression (Promega) by luminometer. p<.05 indicates a significant difference from mock transfected cells and was determined using a student t test with Systat.
8
Profiling ECTV and VACV Induced NF-κB Activation
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HeLa cells or MEF cells were mock-infected or infected with ECTV, VACV, or ECTV-Δ005 at a MOI of 5 for 12 hours followed by stimulation with either 10 ng/ml TNFα (Roche) or 10 ng/ml IL-1β (PeproTech Inc) for 20 minutes. Cells were harvested and lysed in cytoplasmic extract buffer containing 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA (pH 8.0), 0.1 mM EGTA (pH 8.0), 1 mM dithiolthreitol (DTT) and 0.05% NP40. Samples were centrifuged at 1000× g for five minutes to remove nuclei. Supernatants were collected and resuspended in SDS sample buffer. The nuclear pellets were washed and resuspended in nuclear extract buffer containing 20 mM HEPES, 25% glycerol, 0.4M NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), and 1 mM DTT and lysis was performed on ice for 30 minutes. Samples were centrifuged at 1000× g for five minutes. Supernatants were collected as nuclear extracts and mixed with SDS sample buffer.
9
Quantification of IκBα Degradation
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HEK293 cells were plated in 60 mm dishes and transfected with 3 ug pcDNA3.1 – PLpro and 250 ng pIkBα-HA per dish (Mirus LT-1) [42] (link). Cells were incubated for 24 hours, media, removed, and fresh media containing TNFα (Roche) at a final concentration of 20 ng/ml was added and incubated with cells. Cells were incubated with TNFα for the indicated time points. Cells were then lysed with an IkBα lysis buffer: 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 2.5 mM Na pyro-phosphate, 1 mM Beta-glycerophosphate, 1 mM Na ortho-vanadate, 1 ug/ml Leupeptin. Lysates were kept on ice and centrifuged at 14000× g for 10 min at 4C. After centrifugation, supernatant was added to equal volume 2× sample buffer, boiled for 5 min, and then immediately run on 10% SDS-PAGE gel. Gel was transferred to PVDF using a semi-dry blot apparatus (TransBlot Turbo, Bio-Rad) and blocked in 5% milk overnight. Antibodies were used at a concentration of 1∶5000 (mαHA, Covance; mαV5, Invitrogen). Western blots were quantified using a Typhoon Image Scanner and ImageQuant5. p<.05 indicates a significant difference from WT PLpro transfected cells and was determined using a student t test with Systat.
10
Endothelial Cell Response to Shiga Toxin
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The experiments were performed using human endothelial cells obtained from adult renal tissue that were removed due to medical reasons or isolated from human umbilical veins. The described studies were approved by the appropriate ethics committee and were therefore performed in accordance with the ethical standards laid down in the appropriate version of the 1964 Declaration of Helsinki. All donors gave their informed consent prior to inclusion in this study.
Primary HGMVECs were obtained from human kidneys of three donors and cultured as described previously [38 (link)]; passages 8–10 were used for experiments. HUVECs from three donors were harvested according to a previously described method [39 (link)], and passages 3–4 were used for experiments. Confluent cells were stimulated for 6, 12, 24, and 48 h with Stx1 (0.0, 0.1, and 1.0 nM) with or without 24 h preincubation with 10 ng/mL of TNFα (Roche Diagnostics). Stx1 was a kind gift from M. Bielaszewska (University of Münster, Münster, Germany) and was endotoxin-free, as determined by a Limulus assay.
Primary HGMVECs were obtained from human kidneys of three donors and cultured as described previously [38 (link)]; passages 8–10 were used for experiments. HUVECs from three donors were harvested according to a previously described method [39 (link)], and passages 3–4 were used for experiments. Confluent cells were stimulated for 6, 12, 24, and 48 h with Stx1 (0.0, 0.1, and 1.0 nM) with or without 24 h preincubation with 10 ng/mL of TNFα (Roche Diagnostics). Stx1 was a kind gift from M. Bielaszewska (University of Münster, Münster, Germany) and was endotoxin-free, as determined by a Limulus assay.
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