HUVECs were seeded, incubated and analyzed as above for the cell invasion and tubular assays. Groups included (1) Basal medium, (2) NCCM at 10% and 100% doses, (3) CS at 10 and 100 μg/ml and (4) rhNoggin at 10 and 100 ng/ml, with or without recombinant human TNFα (Life Technologies, #PHC3015) (10 ng/ml) including a TNFα control group. For assessment of gene expression, HUVECs from the tubular assay were lysed with Trizol Reagent, RNA extracted (Macherey–Nagel, #740902.50) and cDNA synthesized (Life Technologies, #11754-250) according to manufactures instructions. qRT-PCR was performed using Taqman Gene Expression Assays (Life Technologies, #4331182) for VEGF (hs00900055_m1), MMP-7 (hs01042796_m1), IL-6 (hs00985639_m1), IL-8 (hs00174103_m1) and 18S (hs99999901_s1) using ΔΔCT method36 (link). ΔΔCT are expressed as the mean fold change normalized to the housekeeping gene 18S and either to the Basal or TNFα control, respectively. To verify that an inflammatory microenvironment had been induced by addition of TNFα we performed an enzyme linked immunosorbent assay (ELISA) specific for human TNFα, IL-1β, IL-6, IL-8 (MSD N45025B-1) on Basal and TNFα control media samples. To examine the expression of NGF released into the media a human NGF-beta sandwich ELISA (Peprotech # 900-K60) was also performed on Basal and TNFα control media samples.
Human recombinant tnfα
Manufactured by Thermo Fisher Scientific
Sourced in United States
Human recombinant TNFα is a laboratory reagent produced through recombinant DNA technology. It is a cytokine that plays a role in the inflammatory response. The core function of this product is to serve as a tool for research and scientific investigations, without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Propionate, by Merck Group (2 mentions)
Lps escherichia coli 0111 b4, by Merck Group (2 mentions)
Anti human cd106 vcam 1 pe, by Thermo Fisher Scientific (2 mentions)
Sodium butyrate, by Merck Group (2 mentions)
Egm 2 bulletkit, by Lonza (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Thincert, by Greiner (1 mentions)
Millicell ers 2 voltohmmeter, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Zr 75 30, by American Type Culture Collection (1 mentions)
Rhil 6, by R&D Systems (1 mentions)
Rh ccl2, by R&D Systems (1 mentions)
Rhccl5, by R&D Systems (1 mentions)
Hcc1937, by American Type Culture Collection (1 mentions)
Cama 1, by American Type Culture Collection (1 mentions)
Mda mb 468, by American Type Culture Collection (1 mentions)
Hcc1954, by American Type Culture Collection (1 mentions)
Hek293ft, by Thermo Fisher Scientific (1 mentions)
10 protocols using human recombinant tnfα
1
Inflammatory Effects on Angiogenic Factors
2
Recombinant TNF-α Characterization
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Recombinant human TNF-α was obtained from Life Technologies (Carlsbad, CA, USA). TNF-α was supplied as lyophilized powder and reconstituted as recommended by the supplier. All therapeutic antibody formats were bought from the original provider. All protein concentrations were determined by absorbance measurements at 280 nm using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All solutions for interaction measurements (passivation solution, regeneration solution, buffer solutions) were provided by Dynamic Biosensors GmbH.
3
Caco-2 Cell Barrier Disruption by TNFα
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Caco-2 Cell Culture Caco-2 cells (HTB-37, ATCC Cell Biology Collection, Manassas, VA, USA) were seeded on 24-well semipermeable inserts (0.4 μm, translucent ThinCerts, Greiner Bio-One, Vilvoorde, Belgium) at a density of 10 5 cells per well and cultured for 2 weeks in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal calf serum and 10 mM HEPES buffer (all Life Technologies, Ghent, Belgium). Developing Caco-2 monolayer integrity was monitored by measuring transepithelial electrical resistance (TEER) using a Millicell ERS-2 Voltohmmeter (Merck Millipore, Billerica, MA, USA). Two weeks post seeding, when TEER values of ~700 Ω.cm 2 were obtained, the fully differentiated Caco-2 monolayers were incubated basolaterally with 100 ng/ml recombinant human TNFα (Life Technologies) or an equal volume of medium, and apically with 250 or 500 μM TUDCA (Prodotti Chimici e Alimentari S.p.A., Italy) or an equal volume of medium. Each condition was performed in triplicate. After 48 h, the TEER of the Caco-2 monolayers was measured, cells were collected and total RNA was isolated for quantitative real-time polymerase chain reaction (PCR). Medium from the basolateral compartment was used to determine the concentration of interleukin (IL)-8.
4
Aptamer Inhibition of Tumor Cell Adhesion
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To determine the inhibitory potential of our aptamer on shear-resistant tumor cell adhesion towards human pulmonary endothelium, IBIDI treat microslides VI were coated with confluent HPMEC monolayers, which remained untreated or were stimulated with recombinant human (rh) TNFα (Peprotech) for 4 h prior to the flow adhesion assay (10 ng/mL). The stimulated HPMECs were incubated with 0.15 mg aptamer/mL medium for 30 minutes at 37°C and then perfused with 1×105 HT29 tumor cells per mL as described above (control without aptamer).
5
Therapeutic Antibody Modulation of TNFα Signaling
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Either BATDA-labeled SKBR3 or primary B leukemic cells were left untreated or coated with 10 μg/ml of the respective therapeutic antibodies. Afterwhich, they were pre-incubated in the presence or absence of 10 μg/ml of α-IgG, Fcγ fragment specific (Jackson Immunoresearch) for 30 mins at 37 °C prior to the addition of various concentrations of recombinant human (rh)TNFα (Peprotech) and incubated for 4 hours at 37 °C.
6
Bone Marrow-Derived MSC Characterization
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Primary human bone marrow (BM)-derived MSCs from healthy individuals were purchased from Lonza, grown in MSCGM™ (Lonza GmbH, Cologne, Germany) and used until passage 6 (donor information in Supplemental Materials). Starvation medium was MSCGM™ with all supplements except serum. Cell lines MCF7, T47D, SK-BR-3, MDA-MB-231, MDA-MB-468, MCF10A, THP-1 BT474, HCC1143, HCC1937, HCC1954, CAMA1, ZR-75-30, BT549 were obtained from ATCC (LGC Standards GmbH, Wesel, Germany), HEK293-FT from Invitrogen AG (Invitrogen AG, Carlsbad, USA) (Growth media in Supplemental Materials). All cells lines were authenticated by Multiplexion (Heidelberg, Germany) and negatively tested for mycoplasma contamination.
Recombinant human (rh)-TNFα (PeproTech, RH, USA) and rh-IL-6 (R&D Systems, Wiesbaden-Nordenstadt, Germany) were used at final concentrations of 20ng/ml, rh-CCL2 and rh-CCL5 (R&D Systems, Wiesbaden-Nordenstadt, Germany) at 50ng/ml. IL-6 and CCL5 neutralizing antibodies (NAB) (R&D Systems, Wiesbaden-Nordenstadt, Germany) were used at final concentrations of 2µg/ml, CCL2 NAB at 4.5µg/ml. Protein neutralization was performed for 3h on ice.
Transfections were performed with Lipofectamine® 2000 (Invitrogen AG, Carlsbad, USA) according to manufacturer's instructions. miRNAs and siRNAs were used at final concentrations of 30nM (Details inSupplementary Table 5 ).
Recombinant human (rh)-TNFα (PeproTech, RH, USA) and rh-IL-6 (R&D Systems, Wiesbaden-Nordenstadt, Germany) were used at final concentrations of 20ng/ml, rh-CCL2 and rh-CCL5 (R&D Systems, Wiesbaden-Nordenstadt, Germany) at 50ng/ml. IL-6 and CCL5 neutralizing antibodies (NAB) (R&D Systems, Wiesbaden-Nordenstadt, Germany) were used at final concentrations of 2µg/ml, CCL2 NAB at 4.5µg/ml. Protein neutralization was performed for 3h on ice.
Transfections were performed with Lipofectamine® 2000 (Invitrogen AG, Carlsbad, USA) according to manufacturer's instructions. miRNAs and siRNAs were used at final concentrations of 30nM (Details in
7
Comprehensive Assessment of NF-κB Signaling
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Sodium butyrate, propionate, TSA, WST-1, and a protease inhibitor cocktail were purchased from Sigma-Aldrich. Butyrate and propionate were dissolved in endothelial growth factor (EGM-2) and TSA in DMSO and then further diluted in EGM-2. Concentrations used were based on recent publications of our group [5 (link),6 (link)]. Human recombinant TNFα was bought from eBioscience. The human IL-8 enzyme-linked immunosorbent assay (ELISA) kit, Lipofectamine 2000 (lipo-2000), and the BLOCK-iT Alexa Fluor Red Fluorescent control were purchased from Invitrogen. The NF-κB p65 (pS536) ELISA kit was bought from Cell signaling technology. The IL-33 ELISA kit was purchased from U-CyTech biosciences. The IL-33 monoclonal antibody and Sliencer GAPDH siRNA (human) were bought from Thermo Fisher Scientific. Silencer pre-designed on-Targetplus SMARTpool siRNA IL-33 and siGENOME Non-Targeting siRNA pool (Silencer negative control) were bought from Dharmacon. The following monoclonal antibodies: the anti-phospho p38 (phospho T180 + Y182) antibody, the anti-JNK1+JNK2 (phospho T183 + Y185) antibody, the anti-ERK1/2 (phospho Thr202/Tyr204) antibody, the anti-GAPDH antibody, the rabbit anti-mouse IgG H&L (HRP) conjugated antibody, and the goat anti-rabbit IgG H&L (HRP) conjugated antibody were purchased from Cell Signaling Technology and Abcam.
8
Epigenetic Regulation of Inflammation
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Sodium butyrate, propionate, trichostatin A (TSA, a selective and reversible hydroxamate inhibitor of class I and II HDACs) (Hebbel et al., 2010 (link)), sodium β-hydroxybutyrate (SHB, antagonist for GPR41 receptor) (Kimura et al., 2011 (link)), and LPS (Escherichia coli 0111:B4) were purchased from Sigma-Aldrich, St. Louis, MO, United States. Sodium acetate was bought from Merck Millipore. A cytotoxicity detection kit (lactate dehydrogenase, LDH) was obtained from Roche. Human IL-6, IL-8 ELISA (enzyme-linked immunosorbent assay) kits, and calcein-AM were purchased from Invitrogen. Human recombinant TNFα, anti-human CD106 (VCAM-1) PE, and viability fixable dyes were bought from eBioscience. GLPG0974 (GLPG, antagonist of GPR43 receptor) (Namour et al., 2016 (link)) was obtained from Tocris Bioscience. Primary anti-GPR41 antibody, anti-GPR43 antibody, and an HDAC activity assay kit were purchased from Abcam. EGM-2 Bulletkit was purchased from Lonza (Switzerland).
9
Endothelial Cell Inflammatory Response
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Sodium butyrate, propionate, and LPS (Escherichia coli 0111:B4) were purchased from Sigma-Aldrich, St. Louis, MO, United States. Acetate was bought form Merck Millipore (Germany). Cell cytotoxicity detection kit (lactate dehydrogenase, LDH) was obtained from Roche (Switzerland). Human IL-6 and IL-8 ELISA (enzyme-linked immunosorbent assay) kits were purchased from Invitrogen (Netherlands). Human recombinant TNFα, anti-human CD54 (ICAM-1) PE, anti-human CD106 (VCAM-1) PE and viability fixable dyes were bought from eBioscience (Netherlands). EGM-2 Bulletkit was bought from Lonza (Switzerland).
10
Regulation of HIV-1 LTR Activity
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Stably transfected cells were exposed to human recombinant TNF-α (e-Biosciences, San Jose, CA) at a concentration of 20, 50, 100, 200, or 300 ng/mL. Cells were exposed to cytokine for 24 hours, washed, and subsequently harvested for determination of HIV-1 LTR activity as described above. Separately, stably transfected cell lines were transiently transfected with Tat101 (300 ng) using the Amaxa nucleofector system and Ingenio electroporation solution (Mirus Bio) and harvested after 24 hours. Within the context of Tat, untreated refers to transfection with the parental pcDNA3.1 plasmid without the Tat gene (in other words, empty vector). Independently, cells were also exposed to the HDAC inhibitor trichostatin A (TSA) (400 nM) for 24 hours with or without TNF-α stimulation as indicated above and then LTR-driven GFP transcription was determined using flow cytometry as described above.
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