After 14 days, culture medium was changed for a starvation medium without FCS for 24 hours. Before stimulation, 2-hydroxyquinoline (2HQ), a selective PON inhibitor, was added at the concentration of 100 μM together with 0.1% dimethyl sulfoxide (DMSO) alone or increasing concentrations of 3-oxo-C12 (Sigma-Aldrich®) and 3-oxo-C12:2 in 0.1% DMSO. Caco-2/TC7 cells were stimulated by Interleukin 1 beta (IL-1B) (Sigma-Aldrich®) at 25 ng/mL. After 18 hours, cell supernatants were removed for an Interleukin 8 (IL-8) assay, and cells were washed and scraped into 200 μL Triton 1X. IL-8 concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) (DuoSET Human CxCL8/IL-8, R and D Systems®) and quantified to the total cell-protein content. All experiments were done in duplicate.
Human cxcl8 il 8 duoset
Manufactured by R&D Systems
Sourced in United States, Germany
The Human CXCL8/IL-8 DuoSet is an ELISA development kit for the quantitative measurement of human CXCL8/IL-8 in cell culture supernatants, serum, and plasma samples. The kit includes the necessary components to develop a sandwich ELISA: capture and detection antibodies, and a recombinant protein standard.
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16 protocols using human cxcl8 il 8 duoset
1
Modulation of IL-8 Production by Quorum Sensing Molecules
2
Measurement of IL-8 Secretion in Borrelia-Stimulated Cells
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IL-8 secretion levels were measured in culture supernatants of unstimulated and Borrelia-stimulated cells by ELISA. The protocol was based on sandwich techniques (DuoSet® Human CXCL8/IL-8, Cat# DY208), as described by the manufacturer (R&D systems, Lille, France). Results are representative of three independent experiments.
3
Serum Cytokine Profiling by ELISA
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The concentrations of the cytokines IL-6, IL-8, IL-10, and TNF-α from serum samples collected on days −1, 3, 5, and 13 were determined using commercially available ELISA kits (bovine IL-4, IL-6, or TNF-α screening set [Thermo Scientific]; human CXCL8/IL-8 DuoSet [R & D Systems, Wiesbaden, Germany]; bovine IL-10 ELISA kit [MyBioSource Inc., San Diego, CA, USA]) according to the manufacturer’s instructions.
4
Cytokine Secretion in Cell Cultures
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2,500 cells were plated on each well of a 96-well tissue culture plate (BD) with at least 10 replicates per condition. The medium was left unchanged starting from 48 h after plating or 16 h after transfection. After 48 h, the medium was collected and CXCL9, CXCL11, CXCL8, IL-17F, and TSLP levels were determined using mouse CXCL9/MIG Duoset, human CXCL11/I-TAC Duoset, human CXCL8/IL-8 Duoset, human IL-17F Duoset, and human TSLP Duoset (all from R&D Systems) according to the manufacturer’s protocol. Every experiment was performed at least three times, each with at least three biological replicate samples.
5
Multiplex Cytokine and Chemokine Profiling
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The supernatants were assayed using the following ELISA kits according to manufacturer’s instructions: IL-5 (Human IL-5 DuoSet, range of quantification 23.4–2,000 pg/mL; R&D Systems, Abingdon, UK), CXCL8 (Human CXCL8/IL-8 DuoSet, range of quantification 31.2–2,000 pg/mL; R&D Systems), CCL11 (Human CCL11/Eotaxin Duoset, range of quantification 15.6–1,000 pg/mL; R&D Systems). Supernatants from the second challenge performed on the 12 patients with asthma and allergic rhinitis were also assayed using Mesoscale Sector® (from now on referred to as MS) Imager 6000 (Meso Scale Discovery, Rockville, MD, USA) according to manufacturer’s instructions for the following cytokines: CCL11, CCL26, CXCL10, CCL2, CCL13, CCL22, CCL4, CCL17, INF-γ, IL-1β, IL-2, IL-4, IL-5, CXCL8, IL-10, IL-12p70, IL-13, TNFα (Human TH1/TH2 10-Plex Ultra-Sensitive Kit; Meso Scale Discovery) and CCL11, CCL26, CXCL10, CCL2, CCL13, CCL22, CCL4, CCL17 (Human Chemokine 9-Plex Ultra-Sensitive Kit; Meso Scale Discovery).
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
Quantification of Secreted IL-8 by ELISA
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Quantification of secreted interleukin 8 (IL-8) was conducted using R&D Systems human CXCL8/IL-8 DuoSet enzyme-linked immunosorbent assay per the manufacturer’s instructions (103 (link)).
8
Quantification of Secreted IL-8 by ELISA
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Quantification of secreted interleukin 8 (IL-8) was conducted using R&D Systems human CXCL8/IL-8 DuoSet enzyme-linked immunosorbent assay per the manufacturer’s instructions (103 (link)).
9
Endometrial Epithelial Cell Interactions
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Supernatants of the co-culture of endometrial epithelial cells with isolated lactobacilli and L. vaginalis were analysed for concentrations of IL6 and IL8 by ELISA, as well as of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) by EIA. Concentrations were measured after 24 and 48 h of co-culture using commercially available kits according to the manufacturer’s instructions [Bovine IL-6 Screening Set (Thermo Scientific); Human CXCL8/IL-8 DuoSet (R&D Systems, Wiesbaden, Germany); Prostaglandin F2α EIA Kit (Cayman Chemical, Ann Arbor, USA); Prostaglandin E2 EIA Kit (Cayman Chemical)]. Dilutions of the samples within the detection range were used for each ELISA or EIA. Cross-reaction of the antibody pairs of Human CXCL8/IL-8 DuoSet to bovine IL8 has previously been shown [36 (link)]. Cell culture supernatants were centrifuged twice (400 g and 16,200 g for 5 min, respectively) after harvesting to remove epithelial cells and bacteria. Supernatants were stored at -80°C until measurement. Each experiment was conducted in duplicate using endometrial epithelial cells isolated from three different animals. Concentrations of pro-inflammatory factors in cell culture supernatants were estimated using a microtitre reader (iMark Bio Rad, Bio-Rad Laboratories, Munich, Germany) by comparing with standard curves.
10
Quantification of Inflammatory Cytokines
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Serum concentrations of the inflammatory cytokines interleukin (IL)-6 and IL-8 were quantified using commercial ELISA kits (Human IL-6 Duo Set and Human IL-8/CXCL8 Duo Set, respectively; R&D Systems Inc., Minneapolis, MN, USA), according to the manufacturer’s instructions. The reactions were read at 540 nm or 570 nm using a SpectraMAX 340® microplate reader.
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