For measurements of TNF-α and IL-6 in Mφ culture supernatants, Human TNF-α DuoSet ELISA kits (DY210-05) and Human IL-6 DuoSet ELISA kits (DY206-05) (R&D systems Bio-techne, Abingdon, United Kingdom) were applied and manufacture’s standard protocol was followed.
Human tnf α duoset elisa kit
Manufactured by R&D Systems
Sourced in United States
The Human TNF-α DuoSet ELISA kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human tumor necrosis factor-alpha (TNF-α) levels in cell culture supernatants, serum, and plasma.
Automatically generated - may contain errors
Lab products found in correlation
Human il 6 duoset elisa kit, by R&D Systems (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Ifn γ duoset elisa kit, by R&D Systems (2 mentions)
Human il 6, by R&D Systems (1 mentions)
Fluostar omega fluorescence 556 microplate reader, by BMG Labtech (1 mentions)
Human cell death detection elisaplus, by Roche (1 mentions)
Cobas c501 analyzer, by Roche (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
12 protocols using human tnf α duoset elisa kit
1
Quantifying Cytokine Levels in Macrophages
2
Biomarker Analysis in Rheumatic Disorders
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The BSSL concentration in culture media, plasma and serum was analyzed using an in-house sandwich enzyme-linked immunosorbent assay (ELISA), essentially as previously described [7 (link)]. In this study, the monoclonal mouse anti-human BSSL antibody AS20 (see above) was used as capture antibody and a biotinylated polyclonal rabbit anti-human BSSL antibody as detection antibody. BSSL´s enzymatic activity was determined as the capacity to hydrolyze radiolabeled triglyceride or cholesterol ester substrates as previously described [21 (link), 22 (link)]. The cytokines in RA plasma were measured using Human IL-1β/IL-1F2, Human IL-6 and Human TNF-α DuoSet ELISA kits from R&D Systems (Minneapolis, MN, USA). Assays were performed according to the manufacturer’s protocol and analyzed with a Multiskan GO and ScanIt Software 4.1 for Multiplate Readers (Thermo Fisher Scientific). In PsA patients and healthy controls, hs-CRP and calprotectin (S100A8/A9) were measured in serum whereas cytokines and chemokines were measured in plasma as previously described [28 (link)]. ESR, CRP, Hb, leukocyte-, neutrophil-, and thrombocyte counts, s-urea and s-creatinine were measured using routine methods at the Department of Clinical Chemistry, University Hospital of Umeå, Sweden.
3
ELISA-Based Measurement of TNFα
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ELISAs were conducted as previously described [25 (link)]. Supernatants from healthy and rheumatoid arthritis donor cell cultures treated with PLX51107 (50 nM, 100 nM, 200 nM, 250 nM) or the vehicle, dimethyl sulfoxide (DMSO, 250 nM), were collected after 48 h using the Human TNFα DuoSET ELISA kit (R&D Systems, Minneapolis, MN, USA) utilizing manufacturer provided instructions. To measure FcγR-mediated TNFα production, both healthy- and RA-donor cell cultures were treated with 350 μg/mL heat aggregated IgG (ΔIgG) in the presence of 1 μL of polymyxin B for 24 h.
4
ELISA Analysis of Cytokine Profiles
5
Quantification of T Cell Activation Cytokines
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The concentration of TNF-α and IFN-γ were quantitated in the supernatant collected from the T cell activation assay (the T cell and GBM co-culture +/- CA9 DATE) using commercially available human TNF-α DuoSet ELISA kit (R & D Systems, Cat#DY210-05) and IFN-γ DuoSet ELISA kit (R & D Systems, Cat#DY285B-05) respectively. The sensitivity limits of TNF-α and IFN-γ assay were 15.60 pg/ml and 9.38 pg/ml, respectively. The experiment was performed in duplicates and the OD was measured at 450 nm using the FLUOstar Omega Fluorescence 556 Microplate reader (BMG LABTECH). The IFN-γ concentration in ccRCC model was quantified using the eBioscience Ready-SET-Go human IFN-γ ELISA kit (ThermoFisher, Cat#88-7386-88).
6
Quantification of Circulating Cell Death and Inflammatory Markers
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Histone/DNA complexes in sera and plasma were measured using the Human Cell Death Detection ELISAPLUS (Roche Diagnostics) (24 (link), 38 (link)). The concentrations of NE in sera and plasma were measured by sandwich ELISA, utilizing the Elastase/a1-PI Complex ELISA Kit (Calbiochem) (24 (link), 38 (link)). For the detection of MPO in sera and plasma, the Hycult Biotech ELISA Kit was used (24 (link), 38 (link)). For the detection of A1AT in sera and plasma, the AssayPro ELISA Kit was used. TNF-α concentrations in sera and plasma were assessed by sandwich ELISA with Human TNF-α DuoSet ELISA Kit (R&D Systems).
7
Measuring TNF-α in PCLS Supernatants
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TNF-α levels were measured in PCLS supernatants using a Human TNF-α DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
8
Leishmania braziliensis Infection in Macrophages
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BMDM (106/well) were stimulated with IFN-γ (100 UI/mL) for 24h and infected with stationary-phase Leishmania braziliensis promastigotes for an additional 24h. Macrophages were then washed twice to remove any non-internalized parasites, the RPMI cell medium was replaced and IFN-γ stimulation was reapplied along with the obtained IC50 concentrations of AmB, AmB-D and AmB-NLC, or Ø-NLC, for 48h (Table 1 ). After collecting culture supernatants, the Griess reaction was used to measure nitric oxide (NO) production. Production levels were measured in culture supernatants using a Human TNF-α DuoSet Elisa kit (R&D), in accordance with the manufacturer’s instructions.
AmB-NLC Characterization
| Diameter | PDIª | Zeta Potential | Amphotericin B | EEb |
|---|---|---|---|---|
| 242.0 ± 18.3 nm | 0.44 ± 0.02 | −42.5 ± 1.5 mV | 1.02 ± 0.05 mg mL−1 | ~ 99% |
9
Quantifying TNF-α Levels via ELISA
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TNF-α levels were quantified using a Human TNF-α DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocols.
10
Metabolic Biomarker Evaluation in Fasting Adults
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Weight and height were measured on an auto-anthropometer (super-view, HW-3080, Taiwan) with subjects wearing light clothing, and body mass index (BMI) was calculated by dividing the weight (kg) by the height squared (m2). Blood samples were taken from each subject by peripheral venipuncture after 12 h fasting at the baseline and at 3, 6, and 12 months. Blood samples were separated by centrifugation at 3000 rpm for 10 min at 4 °C to obtain the serum and frozen at −80 °C for further processing. Fasting serum glucose (Glucose AC), ALT, AST, uric acid (UA), creatinine (CRE), blood urea nitrogen (BUN), and albumin (ALB) in the serum were determined with the Cobas C501 analyzer (Roche Ltd., Mannheim, Germany), using commercial kits (Roche Ltd., Germany). Glycated hemoglobin (HbA1c) was measured by high-performance liquid C\chromatography (HPLC) (Trinity Biotech, Iran). For cytokine measurement, a human TNF-α DuoSet ELISA kit (R&D Systems, Minneapolis, MN , USA) was used following the manufacturer’s instructions.
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