An enzyme-linked immunosorbent assay was performed to detect and quantify inflammatory cytokines in supernatants from cocultures of ovarian cancer cell lines and PBMC. ELISA kits were purchased from R&D Systems (Human IL-1β/IL-1F2 DuoSet, Human TGF-β DuoSet, Human TNF-α DuoSet, Human IL-12 p70 DuoSet ELISA) and were used according to the manufacturer’s protocol. The optical density of the colorimetric reaction was determined using an ELISA plate reader at 450 nm. Recombinant human IL-1β, IL-12, TNF-α, and TGF-β were used to establish the standard curve for each assay.
Human tnf α duoset
Manufactured by R&D Systems
Sourced in United States
The Human TNF-α DuoSet by R&D Systems is a kit designed for the quantitative determination of human Tumor Necrosis Factor alpha (TNF-α) levels in cell culture supernates, serum, and plasma samples. The kit includes a capture antibody, detection antibody, and recombinant human TNF-α standard.
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Lab products found in correlation
Human il 6 duoset, by R&D Systems (2 mentions)
Human il 12p70 duoset elisa, by R&D Systems (1 mentions)
Human il 1β il 1f2 duoset, by R&D Systems (1 mentions)
Recombinant human tnf α, by R&D Systems (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Optieia tmb substrate, by BD (1 mentions)
Penicillin g streptomycin sulfate, by Merck Group (1 mentions)
Escherichia coli lipopolysaccharide lps, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline solution, by Merck Group (1 mentions)
Human il 12 p70 duoset, by R&D Systems (1 mentions)
Human il 10 duoset, by R&D Systems (1 mentions)
Human cxcl8 il 8 duoset, by R&D Systems (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Lactate assay kit, by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Lipopolysaccharide lps from e coli, by Merck Group (1 mentions)
Rat tnf α duoset elisa, by R&D Systems (1 mentions)
L6529, by Merck Group (1 mentions)
11 protocols using human tnf α duoset
1
Cytokine Quantification in Ovarian Cancer
2
Modulation of TNF-α Signaling in RASFs
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RASFs were seeded in a 24-well culture plate at an initial density of 5 × 104 cells/well and cultured as described above. After 4 h of pretreatment with bLF (1, 10, and 100 μg/mL), cells were washed with PBS. The cells were then incubated with rhTNF-α (10 ng/mL) for 48 h, and the culture media were collected.
Protein levels in the culture media were measured using an ELISA kit (Human TNF-α DuoSet; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
Protein levels in the culture media were measured using an ELISA kit (Human TNF-α DuoSet; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
3
Innate Immune Response to Rickettsia
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PMA-differentiated THP-1 cells at 5 × 104 THP-1 cells per well were infected with R. conorii or R. montanensis (MOI = 10), and centrifuged at 300 × g for 5 min at room temperature to induce contact. 24 h after infection, 5 μg/mL Escherichia coli O26:B6 Lipopolysaccharide (Invitrogen) in culture media or media alone was added, and incubated for 24 additional hours. 48 total hours after infection, the media was removed, and TNFα concentration was determined by ELISA with Maxisorp plates (Nunc), Human TNFα Duo Set (R&D Systems), and OptiEIA TMB substrate (BD biosciences). Absorbance was measured at 450 mn and standard curve generated with recombinant human TNFα (R&D Systems).
4
Neutrophil Activation by Bacterial Components
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PMNs were resuspended in HBSS containing 1.3 mM CaCl2 and 0.5 mM MgCl2 at a final concentration of 2×106/mL. Cells were incubated with BCL components at 37°C with 5% CO2 for 2–8 hours in 100 µL in 96-well cell culture plates. In the NF-κB inhibition assay, some PMNs were treated with 50 µM of (E)-3-(4-Methylphenylsulfonyl)-2-propenetrile (Bay11-7082, an inhibitor of IκBα phosphorylation, Sigma) for 1 h before addition of BCLs. IL-8 and TNF-α in cell supernatants were measured by ELISA using BD OptEIA human IL-8 ELISA set and human TNF-α DuoSet (R&D Systems) following the manufacturer’s instructions. All samples were assayed in duplicate.
5
Cytokine Release from Epithelial Cells
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The release of TNF‐α and CX3CL1 (fractalkine) into the basolateral medium was analyzed by ELISA using Human TNF‐α DuoSet and Human CX3CL1/Fractalkine DuoSet kits (R&D Systems). For blocking experiments, the soluble TNF‐α receptor fusion protein, Enbrel® (Entanercept) (Wyeth Europa Ltd., Maidenhead, UK) was added to the apical and basolateral medium at a concentration of 10 μg/ml, 1 h prior stimulation.
6
Immune Cell Response to LPS Stimulation
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RPMI 1640 medium and fetal calf serum (FCS) were obtained from Gibco-BRL/Life Technologies, Italy. HEPES buffer, penicillin G-streptomycin sulfate, Dulbecco's phosphate-buffered saline solution (PBS), Histopaque 1077, and Escherichia coli lipopolysaccharide (LPS) were obtained from Sigma-Aldrich Srl, Milan, Italy. The monoclonal antibody Leu-M3 (anti-CD14) was from Becton Dickinson (San Jose, CA). Enzyme-linked immunosorbent assay (ELISA) kits (human TNF-α DuoSet, human IL-6 DuoSet, human IL-12 p70 DuoSet, human CXCL8/IL-8 DuoSet, and human IL-10 DuoSet) were obtained from R&D Systems (Space Srl, Milan, Italy).
7
Monocyte immune response to MV130 and LPS
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Plated human monocytes were stimulated at day 0 with MV130 (2 × 104 bacteria/well) or excipient in a final volume of 200 μL for 24 h, washed with fresh medium and rested. On day 7, cells were washed again and further stimulated with 1 μg/mL LPS for 24 h and supernatants were collected for TNF-α and IL-6 measurement by ELISA, following manufacturer's instructions (Human TNF-α DuoSet and Human IL-6 DuoSet, both from R&D Systems). When required, cells were pre-treated with the epigenetic inhibitors 5′-Deoxy-5′-(methylthio) adenosine (MTA) (1 mM) or pargyline (3 μM) 60 min prior to MV130/excipient stimulation. To explore the metabolic status of the cells, after the challenge with MV130 or excipient, and always prior to LPS stimulation, supernatants were collected at days 1 and 7 and lactate concentration was determined by Lactate Assay Kit (Sigma-Aldrich), following manufacturer's instructions.
8
Quantifying Tumor Necrosis Factor-Alpha
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PCLS culture supernatants were collected, supplemented with 0.2% P1860, and stored at −80 °C. Human Tumor necrosis factor-alpha (TNF-α) was measured using the human TNF-α DuoSet from R&D Systems (Wiesbaden-Nordenstadt, Germany) according to the manufacturer’s specifications and as described elsewhere [7 ]. OD was determined at 450 nm (reference wavelength 540 nm) using the Tecan reader Infinite 200 PRO (Crailsheim, Germany). Cytokine concentration refers to 2 slices per time point and donor, measured in duplicate.
9
LPS-induced TNF-α secretion in macrophages
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Lipopolysaccharide (LPS from E. coli, 100 ng/ml, Sigma Aldrich) stimulation of hMac and rMac to trigger TNF-α secretion was done after 6 d culture in CM. LPS was added without media change. 24 h after LPS stimulation, culture SN was harvested, centrifuged at 420 x g for 10 min and stored in -80°C for further use.
TNF-α levels in hMac and rMac SN were assessed using human TNF-α DuoSet and rat TNF-α DuoSet ELISA (both R&D Systems, Minneapolis, USA), respectively, as per manufacturer’s instructions. The level of TNF-α was normalized to cell confluence (%, measured with a Incucyte SX5) and presented as relative value to LPS-stimulated CTRL.
TNF-α levels in hMac and rMac SN were assessed using human TNF-α DuoSet and rat TNF-α DuoSet ELISA (both R&D Systems, Minneapolis, USA), respectively, as per manufacturer’s instructions. The level of TNF-α was normalized to cell confluence (%, measured with a Incucyte SX5) and presented as relative value to LPS-stimulated CTRL.
10
Whole Blood Stimulation Assay
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Whole blood was diluted 1:10 in serum-free RPMI media (Sigma) containing 5 mM glucose with penicillin (50 U/ml) and streptomycin (50 ug/ml). Diluted whole blood (540 ul) was seeded in 24-well plates (Costar). Whole blood cultures were stimulated with lipopolysaccharide (LPS, from Escherichia coli 055:B5; L6529, Sigma) at 1 or 10 ng/ml with or without 10 ng/ml IL10. Whole blood cultures were incubated at 37 °C in 5% CO2 and supernatants were collected at six hours for analyses of secreted TNF-α on duplicate samples by ELISA (Human TNF-α DuoSet, R&D Systems) according to the manufacturer’s instructions. Absorbance was read at 450 nm on an iMark Microplate Absorbance reader (Biorad, CA, USA) and data were analyzed using Microplate Manager 6.0 (Biorad) to calculate TNF-α concentration using a cubic spline regression model. TNF-α concentration was expressed in pg/ml or as percent of maximal LPS-stimulated secretion.
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