The small intestine of treated and control animals was flushed with 5 ml of 1X PBS pH7.4 (Sigma, St. Louis, MO, United States) and this fluid was centrifuged at 10,000 g at 4°C for 10 min to separate particulate material. The supernatant was collected and kept frozen at −80°C until use. IL-6 and IL-10 were determined in supernatant using the corresponding mouse ELISA Set (BD OptEIA, BD Biosciences PharMingen, San Diego, CA, United States) with catalog numbers respectively: 555240 and 555252. For IL-6, capture antibody was diluted at 1:250 in coating buffer (BD OptEIA, BD Biosciences PharMingen, San Diego, CA, United States) while the detection antibody was diluted at 1:259 in assay diluent (BD OptEIA, BD Biosciences PharMingen, San Diego, CA, United States). For Il-10, dilutions were respectively 1:250 dilution in coating buffer for capture antibody while 1:250 dilution in assay diluent for the detection antibody. For secretory IgA, the level of IgA was analyzed by DAS-ELISA using affinity-purified goat anti-mouse IgA antibodies (α-chain specific) was added at 1.25 mg/well and horseradish-peroxidase conjugated anti-IgA specific antibodies at 1.25 mg/well (Sigma Chemical Co., St Louis, MO, United States). Absorbance was read at 450 nm within 30 minutes of stopping reaction with wave length correction 570 nm.
Opteia
Manufactured by BD
Sourced in United States, Germany, United Kingdom, Belgium, France, Australia, Sweden, Austria
The BD OptEIA is a enzyme-linked immunosorbent assay (ELISA) kit designed for the quantitative detection of specific analytes in biological samples. It provides a standardized and reliable method for the measurement of target molecules without interpretation or extrapolation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Lipopolysaccharides (lps), by Merck Group (5 mentions)
Ready set go, by Thermo Fisher Scientific (3 mentions)
Microplate reader, by Agilent Technologies (3 mentions)
Model 3550, by Bio-Rad (3 mentions)
Duoset elisa, by R&D Systems (3 mentions)
Synergy h4, by Agilent Technologies (3 mentions)
Nunc immuno plate, by Thermo Fisher Scientific (3 mentions)
Multiskan mcc 340, by Thermo Fisher Scientific (2 mentions)
Gm csf, by BioLegend (2 mentions)
Lps ek, by InvivoGen (2 mentions)
Interleukin 4 (il 4), by BioLegend (2 mentions)
Eia vap, by RayBiotech (2 mentions)
Prism 8, by GraphPad (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Duoset elisa development system, by R&D Systems (2 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (2 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (2 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (2 mentions)
Multiskan fc, by Thermo Fisher Scientific (2 mentions)
350 protocols using opteia
1
Intestinal Cytokine and IgA Analysis
2
Cytokine Profiling from Cell Culture
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Cytokines from cell culture supernatants or peritoneal cavity were either
analyzed by commercial available Elisa’s IL-1α
(R&D Systems, sensitivity 2.5 pg/ml), IL-1β
(cell culture supernatants OptEIA, BD Biosciences, limit of detection
15.6 pg/ml; peritoneal lavage Platinum ELISA, eBioscience,
sensitivity 1.2 pg/ml), IL-10 (OptEIA, BD Biosciences, limit of
detection 31.3 pg/ml), PGE2 (R&D Systems,
sensitivity 41.4 pg/ml) or flow cytometry based CBA
Kit’s (IL-4, -5, -6, -13, -21, IFNγ, MCP-1 and TNF)
(limits of detection: IL-4 0.03 pg/ml, IL-5 5 pg/ml,
IL-6 1.4 pg/ml, IL-13 2.4 pg/ml, IL-21
4.8 pg/ml, IFNγ 0.5 pg/ml, TNF
0.9 pg/ml, MCP-1 52.7 pg/ml) from Beckton Dikinson.
analyzed by commercial available Elisa’s IL-1α
(R&D Systems, sensitivity 2.5 pg/ml), IL-1β
(cell culture supernatants OptEIA, BD Biosciences, limit of detection
15.6 pg/ml; peritoneal lavage Platinum ELISA, eBioscience,
sensitivity 1.2 pg/ml), IL-10 (OptEIA, BD Biosciences, limit of
detection 31.3 pg/ml), PGE2 (R&D Systems,
sensitivity 41.4 pg/ml) or flow cytometry based CBA
Kit’s (IL-4, -5, -6, -13, -21, IFNγ, MCP-1 and TNF)
(limits of detection: IL-4 0.03 pg/ml, IL-5 5 pg/ml,
IL-6 1.4 pg/ml, IL-13 2.4 pg/ml, IL-21
4.8 pg/ml, IFNγ 0.5 pg/ml, TNF
0.9 pg/ml, MCP-1 52.7 pg/ml) from Beckton Dikinson.
3
Bone Marrow-Derived Dendritic Cell Culture and Stimulation
4
Generating Bone Marrow-Derived Dendritic Cells
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BMDCs were generated by culturing BM cells from femurs and tibiae in DMEM high glucose medium supplemented with 10% fetal bovine serum, 10 ng/ml GM-CSF (BioLegend, 576306) and 5 ng/ml IL-4 (BioLegend, 574306) for seven days. BMDCs were simulated with ultrapure LPS-EK (Invivogen, tlrl-prklps). Culture medium after stimulation was used for EILSA to detect TNF (BD Biosciences, Opt-EIA, 555268) and IL-6 (BD Biosciences, Opt-EIA, 555240).
5
Cytokine Profiling of Dendritic and T Cells
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For dendritic cells, stage II cultures were stimulated with LPS and ODN1826, and culture supernatants harvested 24 h later. Supernatants were then analyzed either for IL-12p70 via Enzyme-linked immunosorbent assay (ELISA) (OptEIA BD Biosciences, San Diego, CA, USA) or for a set of 97 pro-inflammatory cytokines using mouse antibody cytokine array C6 (RayBiotech, Norcross, GA, USA). Both types of analysis were performed according to manufacturer’s recommendations.
For T cells, splenocytes from immune and control mice were cultured in RPMI base medium and stimulated with recall and control peptides (20 µg/mL) or left unstimulated, and 24 h culture supernatants were harvested and analyzed by ELISA for Interferon-gamma (IFN-γ), IL-2, IL-4 or IL-10 (OptEIA, BD Biosciences, San Diego, CA, USA) according to manufacturer’s recommendations.
The optical density of the ELISA assay was measured via a Bio-Tek ELx800 absorbance reader. The Cytokine Array membrane was visualized using an ImageQuant LAS 4000 mini (GE Healthcare).
For T cells, splenocytes from immune and control mice were cultured in RPMI base medium and stimulated with recall and control peptides (20 µg/mL) or left unstimulated, and 24 h culture supernatants were harvested and analyzed by ELISA for Interferon-gamma (IFN-γ), IL-2, IL-4 or IL-10 (OptEIA, BD Biosciences, San Diego, CA, USA) according to manufacturer’s recommendations.
The optical density of the ELISA assay was measured via a Bio-Tek ELx800 absorbance reader. The Cytokine Array membrane was visualized using an ImageQuant LAS 4000 mini (GE Healthcare).
6
Quantification of IL-1β and IL-8 Levels
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IL-1β and IL-8 were measured from cell culture medium samples using BD OptEIATM assays and following the manufacturer’s protocol (San Diego, CA, USA). Where needed, samples were diluted in Assay Diluent (BD OptEIATM, San Diego, CA, USA). A commercial TMB substrate solution (BD OptEIATM, San Diego, CA, USA) was used for the IL-1β and IL-8 measurements. OD values were measured at a wavelength 450 nm with a reference wavelength of 655 nm using a spectrophotometer (Bio-Rad Model 550 with the Microplate Manager 5.2 programme; Bio-Rad Laboratories, Inc., Hercules, CA, USA)
7
Quantifying Inflammatory Markers in Mice
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Blood was collected from the orbital sinus of each mouse at the end of the experiment. Serum was obtained by centrifugation at 1700× g for 30 min and kept at −70 °C until analysis. The serum levels of total TNF-α, IL-6, and IgE were measured using mouse TNF-α, IL-6, and IgE ELISA kits (BD OptEIATM, BD Science, CA, USA), according to the manufacturer’s instructions. Culture media were obtained approximately 24 h after treatment with EA and stored at −70 °C. The production levels of TNF-α and IL-6 were assessed using EIA kits for humans (BD OptEIATM, BD Science, San Jose, CA, USA) according to the manufacturer’s instructions.
8
Cytokine Quantification in Mice
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Blood was collected from each mouse orbital sinus at the end of the experiment. Serum was obtained by centrifugation at 1700 × g for 30 min and kept at -70°C until analysis. The serum levels of total TNF-α and IL-6 were measured using mouse TNF-α and IL-6 ELISA kits (BD OptEIA TM, BD Science, CA, USA), according to the manufacturer's instructions. Culture media were obtained approximately 24 h after treatment with SF and stored at -70°C. The production levels of TNF-α and IL-6 were assessed using EIA kits for human (BD OptEIA TM, BD Science, CA, USA) according to the manufacturer's instructions.
9
Measurement of IL-6 and IL-8 in Whole Blood
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Whole blood samples were collected from antecubital veins under minimal tourniquet pressure. The plasma levels of IL-6 and IL-8 were determined using high sensitivity enzyme linked immunosorbent assays (ELISA) according to the instructions provided by the manufacturer. The IL-6 ELISA (BD OptEIA, BD Biosciences, San Diego, USA) had a normal range 2.2–12.1 pg/mL and limit of detection of 2.2 pg/mL. For the IL-8 ELISA (BD OptEIA, BD Biosciences, San Diego, USA), the normal range was 10.2–34.3 pg/mL and the limit of detection was 0.8 pg/mL.
10
Cytokine Quantification by ELISA
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The concentrations of cytokines were measured by sandwich ELISA. The IL-6, TNF-α, TGF-β1 (OpTEIA, BD Bioscience, San Diego, CA, USA) and MIF (Duoset R&D Systems, Minneapolis, MN, USA) cytokines were measured according to the manufacturer’s instructions. The concentrations of cytokines in culture supernatants were calculated from a standard curve of each human recombinant cytokine. The limit of detection was according to the last point of the standard curve (IL-6: 4.7 pg/mL; TNF-α: 7.8 pg/mL; TGF-β1: 125.0 pg/mL and MIF 7.8 pg/mL). The data regarding cytokines were normalized by the ratio between the concentration of cytokines in pg/mL and the concentration of total proteins from the Bradford assay in μg/mL, resulting in pg/μg of tissue.
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