HRECs were maintained in Endothelial Cell Basal Medium fortified with EC growth supplements, antibiotics, and fetal bovine serum (Cell biologics, Chicago, IL, USA). Cells were grown on flasks coated with gelatin (0.2%). Cells from passages 6–10 were used for the experiment. All other plastic culture wares, reagents, and chemicals were purchased from Fisher Scientific, Hampton, NH, USA. Considering that the culture medium already contains 5.5 mM glucose, additional D-glucose was added to prepare a total of 30 mM glucose-containing medium for the HG treatment [28 (link)]. The normal medium was served as a control. Primary HRECs were exposed to glucose in two different conditions: (1) by replacing 50 % (50–50) fresh medium each day for 5 d in both the control and treatment groups, and (2) by treating cells once with HG medium with no change for 5 d. BSA-AGE (Cat No. 121800) was purchased from Millipore (Boston, MA, USA). Human TNFα (Cat No. A42550) was purchased from Thermo Scientific (Frederick, MD, USA). LPS lyophilized powder (Cat No. L2018) was obtained from Millipore Sigma (Boston, MA, USA). Triciribine (TCBN) was obtained from Selleckchem (Cat No. S1117), Houston, TX, USA.
Human tnf α
Manufactured by Thermo Fisher Scientific
Sourced in United States, Israel
Human TNF-α is a recombinant protein produced in E. coli. It is a pro-inflammatory cytokine that plays a key role in the immune system and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Human il 1β, by Thermo Fisher Scientific (7 mentions)
Mouse il 1β, by Thermo Fisher Scientific (7 mentions)
Human il 6, by Thermo Fisher Scientific (5 mentions)
Ifn γ, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Human il 17a, by Thermo Fisher Scientific (3 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Il 1β, by R&D Systems (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Murine tnf α, by Thermo Fisher Scientific (2 mentions)
Il 10, by Thermo Fisher Scientific (2 mentions)
Tnf α, by Thermo Fisher Scientific (2 mentions)
Mouse tnf α, by Thermo Fisher Scientific (2 mentions)
Human il 1β, by BioLegend (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Human ifn β, by Thermo Fisher Scientific (2 mentions)
85 protocols using human tnf α
1
Primary HREC Culture and Treatments
2
Cytokine Profiling in Cell Culture
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The level of TNFα, IL-6 and IL-10 was measured in cell culture supernatants using the Human TNFα, Human IL-6 and Human IL-10 Platinum enzyme-linked immunosorbent assay (ELISA) Kits, respectively, according to the manufacturer’s instructions (Thermo Fisher Scientific). The level of IL-1β and IL-18 was measured in cell culture supernatants using the IL1β-IFA-BEST and IL18-IFA-BEST Kits, respectively, according to the manufacturer’s instructions (Vector-best, Russia).
3
Evaluating Mycobacterial Infections in Macrophages
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MAH104 was exposed to the 1 h or the 24 h metal mix, each at corresponding time, while the 7H9 broth exposed bacteria were used as a control. MAH104 of these three phenotypes were used for inoculum preparation. THP-1 macrophages (5 × 105 cells/well) in the 48-well tissue culture plate were infected at MOI of 10 for 1 h, washed twice with HBSS and replenished with fresh RPMI media containing 100 U/mL human TNF-α or 50 U/mL of IFN-γ (ThermoFisher Scientific, Waltham, MA, USA) [24 (link)]. The treatment was repeated at 24 h in fresh RPMI media. The monolayer viability was monitored daily to ensure that there was no toxic effect. Cells were lysed at 48 h post-infection with 0.1% Triton X-100 for 15 min, diluted and plated for quantification of bacterial CFU.
4
Quantification of Th17 Cell Cytokines
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Secreted cytokines from the Th17 cells were quantified in the CM using commercial ELISA kits according to the manufacturer's instructions, human IL-17A (Cat. no. BMS2017, Thermo Fisher Scientific, Inc.), human IL-17F (Cat. no. BMS2037-2, Thermo Fisher Scientific, Inc.), human IL-17A/F (Cat. no. BMS2082, Thermo Fisher Scientific, Inc.), human IL-22 (Cat. no. BMS2047, Thermo Fisher Scientific, Inc.), and human TNFα (Cat. no. BMS223-4, Thermo Fisher Scientific, Inc.). All samples were analyzed as duplicates.
5
Cytokine Secretion and HSF1 Activation
6
Biochemical Assays for Organ-on-Chip Research
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Albumin, urea, LDH and TNF-α were measure as previously reported.17 The albumin ELISA kit was purchased from Bethyl Laboratories (Montgomery TX), the LDH CytoTox 96 kit from ProMega (Madison, WI), urea nitrogen test from StanBio Laboratory (Boerne, TX) and human TNF-α from ThermoFisher Scientific (Waltham, MA). Urea nitrogen, LDH and the TNF-α assays require 10 μL of media each per sample per time point. The assays were conducted following the manufacturer's guidelines with the exception of the urea nitrogen test, which was reconfigured to a 384 well format.17 ,18 The albumin ELISA measurement was determined from 1 μl of media per sample per time point following the manufacture's instructions. All media efflux assay measurements were collected with the SpectraMax M2 (Molecular Devices, Sunnyvale CA) microtiter plate reader. A Data Import Tool (DIT) was used to process raw data from biochemical assays and format the processed data for uploading into the MPS-Db. The DIT is designed to process raw data using a logistic, polynomial or linear regression algorithm to best fit the standard curve for calculating test sample results and format the processed data for uploading into the MPS-Db. For the studies reported here, the logistic regression fit was used.
7
Endothelial Cell Activation by TNF-α
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Human umbilical artery (HUAEC) and vein (HUVEC) endothelial cells were purchased from Sigma (Sigma Aldrich, Merck KGaA, Darmstadt, Germany). The cells were cultured in a Growth Medium MV2 Kit (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) with Microvascular Growth Supplement (MVGS) S00525 (Invitrogen Waltham, MA, USA) in standard culture conditions (Air + 5% CO2, 37 °C) [23 (link)]. Upon confluence, the cells were plated up to the fifth or sixth passage, and then serum-starved (2% MVGS) and exposed to the different experimental conditions. Human TNF-α (Thermo Fisher, Waltham, MA, USA) was diluted in a starving cell medium (2% MVGS) at 50 ng/mL. The cells (HUAEC–HUVEC) were washed three times with sterile PBS 1× buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4). After washing, starved serum (2 mL) and TNF-α (50 ng/mL) were added for 24 h.
8
Caspase-3 and Caspase-8 Activity Assay
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Smac mimetic LCL161, recombinant (active) human caspase-3 (ALX-201-059), substrates Ac-DEVD-MCA (caspase-3), and Ac-IETD-MCA (caspase-8), and inhibitor (zVAD-fmk) were from Sapphire Bioscience (Sapphire Bioscience Pty Ltd). Recombinant (active) caspase-8 was expressed and purified from E. coli using established procedures (41 (link)). Anti-phospho-MLKL (ab196436) antibody was from Abcam, and anti-caspase-8 (1C12, #9746) and anti-cleaved caspase-8 (small subunit, #9748) were from Cell Signaling Technology. Nitrocellulose membrane and ECL Select Western Blotting Detection Reagent were from GE Healthcare. Secondary antibodies were from DAKO (Agilent). All cell culture media: Dulbecco modified Eagle medium, Roswell Park Memorial Institute 1640 (RPMI) medium, Medium 199 (M199), fetal bovine serum (FBS), penicillin/streptomycin (Pen/Strep) and TrypLE Express, and human TNF-α were from Thermo Fisher Scientific. Precast Mini-Protean gels (including TGX Stain-Free) and prestained protein markers were from Bio-Rad Laboratories Inc. All other reagents, including anti-MLKL (MABC604) and anti-β-actin (A5316) antibodies, were from Merck.
9
Quantifying Cytokine Secretion in THP-1 Cells
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IL-6 and TNF-α secretion from THP-1 cells were determined using Human IL-6 DuoSet® (R&D Systems, Minneapolis, MN, USA) and Human TNF-α (Invitrogen, Carlsbad, CA, USA) ELISA kits following the manufacturers’ instructions. Cytokine production was expressed relatively to vehicle treated cells.
10
Quantifying Cytokine Release in THP-1 Cells
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