Firstly, human umbilical vein endothelial cells (HUVECs, the kind gift from the Atherosclerosis Research Centre, Nanjing Medical University, Nanjing, People’s Republic of China) were seeded onto gelatin-coated Transwell polycarbonate filters (0.4 µm pore, 6.5 mm membrane diameter; Corning Incorporated, Corning, NY, USA) and cultured for 3 days to form a restrictive endothelial monolayer. Then, RAW264.7 macrophage cell line (iMΦ) in logarithmic growth phase was plated into the lower compartments of Transwell chambers and co-cultured with HUVECs on the upper compartment of Transwell chambers for a further 24 h. To accelerate endothelial dysfunction, the culture medium was replaced with serum-free M199 containing 10 ng/mL human recombinant tumor necrosis factor-alpha (TNF-α) (PeproTech, Rocky Hill, NJ, USA) and 40 µg/mL oxidized low-density lipoprotein (ox-LDL) (Yiyuan Biotech, Guangzhou, People’s Republic of China).35 (link),36 (link) Finally, the expression of CD44 receptors on HUVEC monolayer and the permeability of HUVEC monolayer after stimulation with TNF-α and ox-LDL for 12 h were analyzed to validate the formation of the injured HUVECs/iMΦ co-culture system.
Recombinant human tumor necrosis factor alpha tnf α
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant human Tumor-Necrosis-Factor-alpha (TNF-α) is a cytokine produced by various cells, including macrophages, lymphocytes, and mast cells. It is involved in the regulation of immune responses, inflammation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Muc5ac, by Cusabio (2 mentions)
Polycarbonate transwell filter, by Corning (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Nci h292, by American Type Culture Collection (1 mentions)
Egm 2mv, by Lonza (1 mentions)
Rottlerin, by Merck Group (1 mentions)
Lipopolysaccharide escherichia coli o111 b4, by Merck Group (1 mentions)
Prostaglandin e2 pge2, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Recombinant human il 1β, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β actin, by Santa Cruz Biotechnology (1 mentions)
Rnaseout ribonuclease inhibitor, by Thermo Fisher Scientific (1 mentions)
Recombinant human interferon ifn γ, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Trizol rna isolation reagent, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
9 protocols using recombinant human tumor necrosis factor alpha tnf α
1
Endothelial Dysfunction in Co-Culture Model
2
NCI-H292 Cell Line Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human lung epithelial cell line, NCI-H292 cell, was purchased from American type culture collection (ATCC; Rockyville, MD, USA). Dexamethasone (DEX) and OVA were purchased from Sigma-Aldrich (St. Louis, MO, USA) and human recombinant tumor necrosis factor alpha (TNF-α) was obtained from Peprotech (Rocky Hill, NJ, USA). The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, IL-5, IL-6, IL-13, and eotaxin (R&D system, Minneapolis, MN, USA), MUC5AC (Cusabio Biotech Co. Wuhan, China) and IgE (BioLegend, CA, USA) were used according to the manufacturer’s instructions. The Diff-Quik® stain kit and Periodic acid-Schiff (PAS) kit was purchased from IMEB Inc. (San Marcos, CA, USA).
3
Airway Inflammation Analysis in NCI-H292 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human lung epithelial cell line, NCI-H292, was purchased from American type culture collection (ATCC; Rockyville, MD, United States). Ovalbumin (OVA) and dexamethasone (DEX) were purchased from Sigma-Aldrich (St. Louis, MO, United States) and human recombinant tumor necrosis factor alpha (TNF-α) was obtained from Peprotech (Rocky Hill, NJ, United States). The enzyme-linked immunosorbent assay (ELISA) kits for IL-4, IL-5, IL-6, IL-13, TNF-α and eotaxin (R&D system, Minneapolis, MN, United States), MUC5AC (Cusabio Biotech Co., Wuhan, China) and IgE (BioLegend, CA, United States) were used according to the manufacturer’s instructions. The Diff-Quik® stain kit and Periodic acid-Schiff (PAS) kit were purchased from IMEB (San Marcos, CA, United States).
4
Cytokine-Induced Endothelial Cell Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the treatment with cytokines, two distinct BEC populations (CD31+PDP−CD157+ and CD31+PDP−CD157−) as well as two distinct LEC populations (CD31+PDP+CD157+ and CD31+PDP+CD157−) were separated by sorting and used at passage 0–1. The cells were seeded into 6-well plates coated with 0.01% gelatin solution and cultured in EGM-2MV (Lonza, Basel, Switzerland) at 37 °C until reaching 80–90% confluency. Two distinct cytokines, recombinant human Tumor-Necrosis-Factor-alpha (TNF-α; Peprotech, Hamburg, Germany) applied at concentration 16 ng/mL, and recombinant human Interferon-gamma (IFN-γ; Peprotech, Hamburg, Germany) applied at concentration 5 ng/mL, were diluted in 2 mL of culture media, added and the cells, which were further cultivated in the incubator (37 °C, 5% CO2) for either 24 h, 48 h, or 72 h. At the mentioned time points, the treatment was stopped by removing the cytokine-media mixture and the treated cells were then used either for flow cytometric analysis or immunofluorescence staining.
5
Macrophage Differentiation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PMA was purchased from Sigma (#P8139). Recombinant human IFNγ was purchased from
BD Biosciences (#554617). Lipopolysaccharide Escherichia coliO111:B4 was purchased from Sigma (#L2630). Rottlerin was purchased from Sigma
(#R5648). Recombinant human granulocyte-macrophage colony-stimulating factor
(GM-CSF) was purchased from Sanofi (Leukine sargramostim). Recombinant human
macrophage colony-stimulating factor 1 (M-CSF) was purchased from GenScript
(#Z02001). Recombinant human interleukin-4 (IL-4) was produced at the Ludwig
Cancer Research (Brussels, Belgium). Recombinant human IL-10 was purchased from
R&D (#1064-IL). Ribomunyl was purchased from Pierre Fabre (France).
Prostaglandin E2 (PGE2) was purchased from Sigma. Recombinant human tumor
necrosis factor alpha (TNFα) was purchased from PeproTech (#300-01A).
Recombinant human transforming growth factor beta (TGFβ) was purchased from
R&D (#240-B).
BD Biosciences (#554617). Lipopolysaccharide Escherichia coliO111:B4 was purchased from Sigma (#L2630). Rottlerin was purchased from Sigma
(#R5648). Recombinant human granulocyte-macrophage colony-stimulating factor
(GM-CSF) was purchased from Sanofi (Leukine sargramostim). Recombinant human
macrophage colony-stimulating factor 1 (M-CSF) was purchased from GenScript
(#Z02001). Recombinant human interleukin-4 (IL-4) was produced at the Ludwig
Cancer Research (Brussels, Belgium). Recombinant human IL-10 was purchased from
R&D (#1064-IL). Ribomunyl was purchased from Pierre Fabre (France).
Prostaglandin E2 (PGE2) was purchased from Sigma. Recombinant human tumor
necrosis factor alpha (TNFα) was purchased from PeproTech (#300-01A).
Recombinant human transforming growth factor beta (TGFβ) was purchased from
R&D (#240-B).
6
Cytokine Inhibition in Cell Model
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The drugs and reagents used in this study comprised: Inhibitor of NF-κ kinase subunit beta (IKKβ) inhibitor ML120B (MedChemExpress, Monmouth Junction, NJ, USA), anti-human IL6 neutralizing antibody (R&D Systems, Minneapolis, MN, USA), anti-human IL1β neutralizing antibody (Abcam, Cambridge, MA, USA), recombinant human IL1β (Peprotech, Rocky Hill, NJ, USA), and recombinant human tumor necrosis factor alpha (TNFα) (Peprotech).
7
Cell Culture and Biochemical Assays for Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plastic for cell cultures and disposable filtration units were purchase from Corning (Milan, Italy). Dulbecco-modified eagle medium (DMEM)/Ham’s F-12 medium (ratio 1:1) with and without phenol red, RPMI-1640 Medium, phosphate-buffered saline Ca2+/Mg2+-free (PBS), bovine serum albumin (BSA) fraction V, antibiotics, EDTA-trypsin solution, Bradford reagent were from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). Recombinant human interferon (IFN-γ) and recombinant human Tumor Necrosis Factor alpha (TNFα) were from Peprotech® (RockyHill, NJ, USA). For RNA extraction, TRIzol RNA isolation reagent was purchased by Ambion™; for reverse transcription 10 mM dNTP mix, random primers, RNaseOUT™ Ribonuclease inhibitor and SuperScript® III Reverse were purchased from Invitrogen. SYBR® Green PCR Master Mix for qPCR was from Life Technologies™ (Applied Biosystems®). All reagents for SDS-PAGE were from Millipore (Billerica, MA, USA). l -Glutamine was from Gibco Laboratories (Grand Island, NY). Polyclonal rabbit anti-PDE5 and monoclonal mouse anti-β-actin were from Santa Cruz (CA, USA).
8
Optimized Chromatographic Purification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco’s modified Eagle medium (DMEM) (4.5 g/L glucose), fetal bovine serum (FBS), and molecular weight protein markers were obtained from Thermo Fischer Scientific (GmbH, Frankfurt, Germany). Recombinant Human Tumor Necrosis Factor alpha (TNFα) was obtained from Peprotech (London, UK). A cocktail of protease inhibitors was obtained from Roche (Mannheim, Germany). Reporter lysis buffer and luciferin were purchased from Promega Corporation (Madison, WI, USA). For chromatography studies, silica gel 60, Celite® 545 particle size 0.02–0.1 mm, CAS 68855-54-9, pH 10 (100 g/L, H2O, 20 °C), neutral alumina Alugram®, and RP C-18 silica gel were used and obtained from Macherey-Nagel (Düren, Germany). 1 H (400 MHz) spectra were measured on Bruker 400 NMR spectrometers. Chemical shifts were referenced to the residual solvent signal (CDCl3: δH = 7.26). Purifications were monitored by TLC on Merck 60 F254 (0.25 mm) plates, visualized by staining with 5% H2SO4 in EtOH and heating. HPLC JASCO Hichrom, 250 × 25 mm, silica UV−vis detector-2075 plus (Oklahoma, Japan) was used. Dexamethasone (DEX) and all the other solvents and chemicals used were obtained from Sigma-Aldrich (St. Louis, MO, USA). Finally, the Chios Mastiha was a kind donation from the Mastiha Shop and the Chios Mastic Growers Association.
9
Evaluating Immune Responses to Tumor Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPMI-1640, HEPES, penicillin/streptomycin (P/S), H 2 O 2 , Viscum album agglutinin-I (VAA-I), lipopolysaccharides (LPS) from Escherichia coli, trypan blue, and dextran were purchased from Sigma-Aldrich Ltd. (Saint-Louis, Missouri); recombinant human tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) were purchased from PeproTech Inc. (Rocky Hill, NJ, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!