TNFα was quantified by using mouse Duoset TNFα ELISA kit (R&D Systems, Minneapolis, MN). Plates were prepared and assayed according to the manufacturer’s protocol. The samples absorbance was analyzed at 450 nm. Lactate dehydrogenase (LDH) release into the conditioned medium was measured using a CytoTox 96 nonradioactive cytotoxicity assay according to the manufacturer’s instructions (Promega, Madison, WI).
Tnfα duoset elisa kit
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany
The TNFα DuoSet ELISA kit is a laboratory product designed for the quantitative detection of human Tumor Necrosis Factor alpha (TNFα) in various sample types. It is an enzyme-linked immunosorbent assay (ELISA) kit that provides the necessary components to measure the concentration of the TNFα protein in a sample.
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Lab products found in correlation
Il 1β duoset elisa kit, by R&D Systems (3 mentions)
Il 6 duoset elisa kit, by R&D Systems (2 mentions)
Ifn γ duoset elisa kit, by R&D Systems (2 mentions)
Z wehd fmk, by R&D Systems (2 mentions)
Caspase 1 mouse matched pair detection set, by Adipogen (2 mentions)
Cathepsin b inhibitor ca 074 me, by Enzo Life Sciences (2 mentions)
Inject alum, by Thermo Fisher Scientific (2 mentions)
Lps e coli strain 055 b5, by Merck Group (2 mentions)
Streptolysin o slo, by Merck Group (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (1 mentions)
Wright giemsa, by Merck Group (1 mentions)
May grunwald, by Merck Group (1 mentions)
Anti inos ab3523, by Abcam (1 mentions)
Vectastain abc kit for immunohistochemistry, by Vector Laboratories (1 mentions)
Anti tnf α ab6671, by Abcam (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Microtainer, by BD (1 mentions)
Ifn β duoset elisa kit, by R&D Systems (1 mentions)
Cd62l, by R&D Systems (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
15 protocols using tnfα duoset elisa kit
1
TNFα and LDH Quantification
2
Antibody-based Characterization of Macrophage Phenotypes
3
Quantification of Gut Inflammation Markers
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Fecal samples were prepared as described previously.8 (link) In brief, frozen or freshly collected feces were dissolved to make a 100 mg/mL fecal suspension in PBS containing 0.1% Tween 20 and vortexed for 30 minutes at room temperature. Samples then were centrifuged at 4°C for 10,000 × g for 10 minutes to collect clear supernatants. Blood was collected at the time of mice euthanasia in a BD microtainer (Becton Dickinson) via retro-orbital plexus. To measure colonic tumor necrosis factor (TNF)α, colonic tissue was homogenized in RIPA buffer and the clear supernatant was used to measure TNFα. Hemolysis-free sera was obtained after centrifugation and stored at -80°C until further use. Mouse Lcn2 and TNFα Duoset ELISA kit (R&D Systems) were used to quantify serum and fecal Lcn2 and colonic TNFα per the manufacturer’s protocol. Likewise, serum amyloid A was quantified in serum using an ELISA kit. Serum immunoreactivity to LPS and flagellin was analyzed as described earlier.44 (link)
4
Quantifying Soluble TNFα Secretion from Microglia
5
Comprehensive Cytokine Profiling in Mouse Tissues
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Mouse spleen, hindlimb muscle (including quadriceps, tibialis anterior, gastrocnemius, and soleus muscles), cardiac muscle, serum, and white adipose tissue were collected. Tissues were homogenized using a Dounce homogenizer (100 strokes per tissue sample) on ice in prechilled buffer containing 100 mM tris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, and proteinase inhibitor. The tissue lysate was centrifuged for 10 min at 13,000 rpm at 4°C. The tissue lysate supernatants, together with the mouse serum collected using sodium citrate tubes, were analyzed using the mouse IL-15/IL-15Rα uncoated ELISA Kit (88-7215-22, Life Technologies), IL-12 p40 DuoSet ELISA Kit (DY2398-05, R&D Systems), IFN-β DuoSet ELISA Kit (DY8234-05, R&D Systems), IFN-γ DuoSet ELISA Kit (DY485-05, R&D Systems), IL-6 DuoSet ELISA Kit (DY406-05, R&D Systems), and TNFα DuoSet ELISA Kit (DY410-05, R&D Systems) following the manufacturer’s instructions. The protein levels in the tissues and serum were normalized to the tissue weight and serum volume, respectively. Where indicated, IL-12 p40 in cell culture supernatants was analyzed and normalized to the protein levels of the cell pellets.
6
Activation and Evaluation of CD8+ T cells
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PBMC from de-identified healthy donors were obtained with informed consent from the Department of Transfusion Medicine, National Institutes of Health under Biological reagent Registry RD-14-IX-12 approved by NIH biological safety board. CD8-depleted PBMCs, either stimulated or non-stimulated with 10 μg/mL phytohaemagglutinin (PHA) or anti-CD3 antibody (with or without conjugated beads), were plated at 2×106 cells/ml in 48 well plates in RPMI 1640 + 10%FBS (R10) to a final volume of 1 mL in each well. IL-2 was added at a final concentration of 20 U/mL. CD3 antibody okt3 was used at ~0.2 μg/mL final concentration. Compounds were prepared in appropriate solvents according to the manufacturer’s instructions, diluted to working concentrations using RPMI 1640 + 10%FBS (R10), and added to the cell cultures for 6–24 h for CD62L shedding, and 48 h for TNF-α release. Supernatants were collected and assessed using either the CD62L or TNF-α Duo Set ELISA kit according to manufacturer’s instructions (R&D Systems, Inc.). The viability of cells treated with compounds was assessed by acridine orange / propidiom iodide (AO/PI Logosbio catalog F23001) staining using Luna-FL cell counter following the manufacture’s instruction.2
7
Quantifying Porcine Cytokine Levels
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Levels of TNF-α and IL-1β in cell culture supernatants were measured by ELISA using the porcine TNF-α DuoSet ELISA kit and IL-β DuoSet ELISA kit (R&D Systems) following the manufacturer’s recommendation. Samples were diluted 1:3 with assay sample diluent and optical density was measured at 450 nm and 540 nm using a BioTek Synergy HTX plate reader (BioTek). Concentrations were calculated as described by the manufacturer using a recombinant protein as standard. Cytokine levels from the priming supernatants were reported as ng/ml of indicated cytokine. Responses following heterologous re-stimulation were analyzed by calculating the fold change of primed samples divided by the matched unprimed (NS/LPS) samples.
8
TNFα Secretion Assay in THP-1 and RAW Cells
9
Cytokine Measurement in Virus-Infected Cells
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Supernatants were collected from cells treated with vehicle or LCL161 and infected with indicated MOI. Cytokines were measured using the TNF-α DuoSet ELISA kit (R&D Systems) or Verikine IFNβ ELISA Kit (PBL Interferon Source). Serum was collected from 5-week-old BALB/c mice infected with 1 × 108 plaque forming units (PFU) virus, and TNF-α levels were measured using the TNF-α Quantikine high-sensitivity assay kit (R&D Systems).
10
Macrophage Cytokine Responses to Uropathogenic E. coli
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BMDMs were incubated with UTI89, CFT073, or MG1655 for 2 hours, then treated with 100 μg/ml gentamicin containing medium for 1 hour and finally incubated with 10 μg/ml gentamicin containing medium for 5 hours (8-hr samples) or 21 hours (24-hr samples) at which point the supernatant was collected for ELISAs. IL-1β DuoSet ELISA kit (R&D systems, Minneapolis, MN), TNFα DuoSet ELISA kit (R&D systems) and Caspase-1 (mouse) matched pair detection set (Adipogen, San Diego, CA) were used according to manufacturer instructions. Where indicated, the following inhibitors were used throughout the experiment: the Cathepsin B inhibitor CA-074 Me (Enzo Life Sciences, Inc., Farmingdale, NY) was used at 10 μM, the caspase-1 inhibitor Z WEHD-FMK (R&D systems) was used at 10 μM, and the VPS34 inhibitor 3-MA (Calbiochem, Millipore, Billerica, MA) was used at 2, 5 and 10 mM. Alum- and Silica-treated samples were pretreated with 100 ng/ml LPS(E. coli strain 055:B5, Sigma) for 2 hours, and then treated with 200 μg/ml of Inject Alum (Thermo Scientific, Rockford, IL) or 100 μg/ml of Silica (Thermo Scientific) in fresh media for 6 hours. Streptolysin O (SLO) (Sigma) was used at 10 μg/ml with 10 mM DTT for 5 min, and then the medium was changed and collected 6 hours later.
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