Apoptosis was induced by replacing the growth medium with medium containing STS, ActD, or human TNF-α added together with CHX (each purchased from Sigma-Aldrich) at the indicated concentrations. Necroptosis was induced by replacing the growth medium with medium containing TSZ (i.e., 20 ng/ml human TNF-α (Sigma-Aldrich), 1 µM Smac mimetic BV6 (Tocris), and 50 µM Z-VAD-FMK (Tocris)). In control wells, DMSO was added instead of pro-death drugs. Treated cells were incubated in presence of pro-death drugs until the end of the monitoring period (time-lapse microscopy) or the time point of analysis (all other experiments). When indicated, cell death inhibitors (necrosulfonamide (Tocris), necrostatin-1 (Tocris), GSK′872 (Merck), Z-VAD-FMK (Tocris), or inhibitors from the caspase inhibitor set IV (Enzo)) or antibiotics (chloramphenicol, tetracycline, and penicillin G (each purchased from Sigma-Aldrich)) were added at the indicated concentrations prior to cell death induction.
Human tnf α
Manufactured by Merck Group
Sourced in United States, Macao
Human TNF-α is a recombinant protein that represents the soluble form of the human Tumor Necrosis Factor alpha (TNF-α) cytokine. TNF-α is a key mediator of inflammation and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (2 mentions)
Tnf α, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Normal goat serum (ngs), by Vector Laboratories (2 mentions)
Rat tnf α, by Merck Group (2 mentions)
Infinite 200 pro plate reader, by Tecan (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Human ifn γ, by R&D Systems (2 mentions)
Gsk 872, by Merck Group (1 mentions)
Penicillin g, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Necrosulfonamide, by Bio-Techne (1 mentions)
Z vad fmk, by Bio-Techne (1 mentions)
Necrostatin 1, by Bio-Techne (1 mentions)
Mouse tnf α, by Thermo Fisher Scientific (1 mentions)
Anti ripk1 antibody, by BD (1 mentions)
Konelab 20i chemistry analyzer, by Thermo Fisher Scientific (1 mentions)
29 protocols using human tnf α
1
Induction and Inhibition of Cell Death Pathways
2
Necroptosis pathway regulation assay
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The following chemicals were purchased: human TNFα (Merck Millipore); mouse TNFα (PeproTech); GSK’872, NSA, Nec1-s (Calbiochem); Lipofectamine 2000, TRAIL (Invitrogen); Nec1, etoposide, tunicamycin, (Sigma-Aldrich). Smac mimetic SM-164 was synthesized by Dr. Shaomeng Wang (University of Michigan). Gi was synthesized as described in Hua Z et al.26 . The antibodies used for the western blot analysis are: anti-RIPK3 (E1Z1D), anti- RIPK1 (p-Ser166), anti- RIPK3 (p-Ser227), anti-caspase-8, anti-His antibodies (Cell Signaling); anti-CK1γ2, anti-MLKL, anti-caspase-3, anti-vimentin antibodies (Genetex); anti- PARP-1, anti-GST, anti-actin, anti-tubulin, anti-calnexin antibodies (Santa Cruz Biotechnology); anti-FLAG, anti-phospho-serine, anti-phospho-threonine antibodies (Sigma-Aldrich); anti-CK1γ1, anti-phospho-MLKL (p-Ser358) antibodies (Abcam); anti-RIPK1 antibody (BD Biosciences) and anti-CK1γ3 (Thermo Fisher Scientific). For the in vitro experiment, GST-RIPK3, GST-CK1γ1 (Sigma-Aldrich), GST-CK1γ3 (Abcam) and His-MLKL (Aviva Systems Biology) proteins were utilized.
3
Fasting Blood Biomarkers in Humans
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In the detailed explanation of study purposes, participants were informed to have not eaten at least for 8 h before blood collection. Additionally, participants were reminded 1 week before the study visit. Venous blood samples were collected at T0 and T1, and in order to obtain serum, samples were centrifuged as previously reported (21 (link)). ESR was measured by Wintrobe method. Serum C-reactive protein (CRP) levels were detected by immunonephelometry (GOLDSITE Diagnostics, Inc., Shenzhen, China). Serum ferritin levels were measured by enzyme-linked immunosorbent assay (ELISA) (Monobind Inc., Lake Forest, CA, United States), with a detection sensitivity limit of 0.17 ng/ml. Serum iron was determined on a Konelab 20i Chemistry Analyzer (Thermo Electron Corporation, Vantaa, Finland) according to the standardized procedures (Method Iron “Ferene S,” Sclavo Diagnostics, Siena, Italy). Quantification of interleukins was performed using ELISA kits for human IL-6, human TNF-α, human IL-1β, and human IL-10 (Merck Life Sciences, Darmstadt, Germany). The respective sensitivities of these assays were 1.6 pg/ml for IL-6, 0.2 pg/ml for TNF-α, 0.2 pg/ml for IL-1β, and 2 pg/ml for IL-10.
4
Isolation and Treatment of Human Gingival Fibroblasts
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Human GF were isolated from gingival papillary explants obtained from clinically healthy donors with no systemic and/or periodontal disease who were informed of the purpose of this study. Briefly, after dissection of gingival biopsies using dispase (Gibco BRL, Grand Island, NY, USA), the epithelial cell layer was microscopically dissected from the underlying connective tissue, and GF was extracted from the subepithelial tissue as previously described.25 (link) Isolated primary human GF were treated with the indicated amounts of LPS derived from P. gingivalis (Invivogen, San Diego, CA, USA) in addition to recombinant human NAMPT protein (AdipoGen, San Diego, CA, USA), human IL-1β (GeneScript, Piscataway, NJ, USA) and human TNF-α (Merck-Millipore, Billerica, MA, USA). Primary cultured human GF were infected with empty (Ad-C), Nampt-expressing adenovirus (Ad-Nampt), or SIRT2-expressing adenovirus (Ad-SIRT2) for 2 h at the indicated multiplicity of infection and incubated for an additional 24 h. The inhibitors FK866 (Cayman, Ann Arbor, MI, USA), an iNAMPT inhibitor; anti-NAMPT antibody (Bio Vision, Milpitas, CA, USA), an eNAMPT inhibitor; NIC (Sigma-Aldrich), a total SIRT inhibitor; and EX-527 (Sigma-Aldrich), a SIRT1 inhibitor, were used at the indicated concentrations in the presence of IL-1β or Ad-Nampt. Dimethyl sulfoxide or phosphate-buffered saline was used as a vehicle.
5
Neutrophil Apoptosis and NET Induction
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Neutrophils were isolated from healthy human donors by density centrifugation at 500g for 30 minutes at 20°C using Polymorphprep (Axis-Shield), and were suspended in RPMI 1640 (Sigma-Aldrich). Apoptosis was induced using 0.05 μM STS (FUJIFILM Wako) for 4 hours. Cell apoptosis was assessed by fluorescence microscopy using TUNEL staining kit (Medical & Biological Laboratories [MBL]). For ANCA-NET induction, neutrophils were primed with human TNF-α (5 ng/mL; Merck) for 15 minutes and then exposed to 400 μg IgGs eluted from the serum of healthy volunteers or patients with MPO-ANCA+ AAV for 4 hours (2 hours for the efferocytosis assay). For PMA-NET induction, neutrophils were exposed to PMA (50 nmol/L; Merck) for 2 hours. NET formation was quantified based on the SYTOX Green+ area of immunostaining as the mean luminance value using ImageJ software. To assess the effects of CD47 blockade on NET induction, neutrophils were exposed to PBS, mouse IgG1 isotype CT-Ab (10 μg/mL; MOPC-21, BioXcell), or anti-CD47 mAb (10 μg/mL; B6H12, BioXcell) for 30 minutes before NET induction.
6
Vasorin Inhibits TNF-α Induced Apoptosis in HTM Cells
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To determine whether vasorin inhibits TNF‐α induced apoptosis in HTM cells, confluent HTM cultures grown on glass coverslips were treated with 30 ng/ml human TNF‐α (Sigma‐Aldrich, St. Louis, MO), and 1 µg/ml cycloheximide (Sigma‐Aldrich, St. Louis, MO. USA) with or without 50 ng/ml vasorin for 24 hrs as described Choksi et al.21 Live cells were then briefly washed with 1X PBS and incubated with propidium iodide (PI), 0.5 µg/ml in serum‐free DMEM media for 5 min at 37°C. PI is a membrane impermeant dye that is generally excluded from viable cells, while intercalating with double‐stranded DNA in cells undergoing apoptosis. Images of red fluorescent positive cells were captured under a fluorescent microscope (10×, Zeiss Axioplan 2). A minimum of 10 images were captured at different locations on the same coverslip, and the number of fluorescent positive cells per unit area was estimated and plotted. Phase contrast images of TM cells exposed to the above treatments were captured using a Zeiss Axiovision microscope.
7
Molecular Regulators of Cell-Cell Interactions
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Human TNF-α, phenylmethylsulfonyl fluoride (PMSF), dimethyloxalylglycine, and triton X-100 were purchased from Sigma (St. Louis, MO). BIX01294 was obtained from Axon Medchem (Groningen, The Netherlands). GSK126 and ML324 were from Cayman (Ann Arbor, MI). Normal goat serum and mounting medium were obtained from Vector Laboratories Inc. (Burlingame, CA). Alexa Fluor® 594 goat-anti-mouse IgG and Alexa Fluor® 488 goat-anti-rabbit IgG were purchased from Molecular probes (Eugene, OR). Anti-ICAM1 mouse monoclonal and rabbit polyclonal antibodies were from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA). Anti-G9a and anti-KDM4B rabbit polyclonal antibodies were from Cell signaling (Danvers, MA). Ant-laminin rabbit polyclonal, anti-β-actin mouse monoclonal, and anti-VCAM1 rabbit monoclonal antibodies were from Abcam (Cambridge, UK). Culture media, M199 and RPMI 1640, fetal bovine serum (FBS), and antibiotics were purchased from Gibco (Carlsbad, CA). MSCVhygro-F-G9a was a gift from Kai Ge (Addgene plasmid # 41721).
8
Cytokine and HIF-1α Inhibitor Study
9
EPC Culture and TNF-α Treatment
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EPCs were cultured on 1% fibronectin coated dishes in EGM-2 Bullet Kit with 15% fetal bovine serum (FBS) known as EGM-2 growth medium (Lonza, VIC, Australia). EPCs were cultured under standard cell culture conditions using a 5% CO2 incubator at 37°C. Tumor necrosis factor (TNF-α) is well known to induce Nox isoforms and cell death. In some conditions, cells were treated with human TNF-α (20 ng/ml; Sigma, St. Louis, MO) for 6 or 48 h prior to cell harvest.
10
HUVEC Stimulation with Glucose, Palmitate, TNF-α, and CoCl2
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HUVECs obtained from umbilical cords were digested with 0.05% type II collagenase (Sigma Chemical Co., St. Louis, MO, USA). Isolated HUVECs were cultured in 20% fetal bovine serum (FBS) (Gibco, Grand Island, USA) in Clonetics TM endothelial cell systems (Lonza, Walkersville, USA) until confluent. Passages 3-5 were starved with 2% FBS in culture medium for 16-24 h before the experiments. For Western blot analysis, HUVECs were divided into two groups: the normal glucose group (cells were cultured in EGM-2 medium containing 5.5 mM glucose), and the high glucose group (glucose was added to the medium for a final concentration of 30 mM). Then, cells were stimulated with or without 200 μM palmitate, 10 ng/ml human TNF-α, or 200 μM CoCl2 (Sigma) for 12 h. Then, HUVECs on dishes were washed with PBS, scraped with lysis buffer, incubated on ice for at least 30 min, and centrifuged at 14,000 rpm for 30 min. The supernatants were stored as cellular lysates at −80°C before analysis.
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