Supernatants from the survival assays were collected to evaluate IL-8 release by INT-407 cells after exposure to the total OMC and OMC fractions.26 (link) IL-8 production was determined using a human CXCL8/IL-8 ELISA Kit (R&D Systems) using standard curves following the manufacturer's instructions. INT-407 cells were exposed to either C. jejuni (MOI 100:1) for 3 h, to phorbol 12-myristate 13-acetate (PMA) for 1 h as a positive control or to medium alone as a baseline; the three treatments were compared.
Human cxcl8 il 8 elisa kit
Manufactured by R&D Systems
Sourced in United States
The Human CXCL8/IL-8 ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human CXCL8/IL-8 in cell culture supernatants, cell lysates, serum, and plasma.
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7 protocols using human cxcl8 il 8 elisa kit
1
Quantifying IL-8 Release in INT-407 Cells
2
Isolation and Quantification of C. jejuni OMC
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All chemical reagents used in this study were of high-grade purity from Sigma-Aldrich, St. Louis, MO, USA, unless otherwise specified. Endotoxin-free sterile water (Baxter Healthcare Corporation, Chicago, IL, USA) was used for the dialysis of all C. jejuni OMC fractions. Dulbecco's phosphate-buffered saline (DPBS) without CaCl2 and MgCl2 (Invitrogen, Grand Island, NY, USA) was used in all experiments. A bicinchoninic acid (BCA) Kit (Pierce, Rockford, IL, USA) was used for protein estimation, and a human CXCL8/IL-8 ELISA Kit was used for IL-8 quantification (R&D Systems, Minneapolis, MN, USA).
3
Cytokine modulation by Lactobacillus
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Supernatants from co-culture assays were tested for the effects of soluble factors produced by Lactobacillus on IL-8 production and other related cytokines. IL-8 concentrations in LCM-treated, C. difficile-stimulated HT29 or Caco-2 cell co-culture supernatants were measured using a Human CXCL8/IL-8 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. To determine whether Lactobacillus spp. can modulate cytokines other than IL-8 in this assay, cell supernatants from C. difficile-stimulated HT-29 cells in the presence or absence of LCM were screened by a Human Cytokine/Chemokine-Premixed 14-plex kit (Millipore, Billerica, MA) in a Luminex 100 system (Luminex Corporation, Austin, TX) for quantification of the analytes GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, MCP-1, and TNF-α. Analytes at concentrations exceeding the minimum detectable dose were evaluated and raw data were obtained with MasterPlex CT version 1.2.0.7 and analyzed with MasterPlex QT version 5.0.0.73 (Hitachi MiraiBio, San Francisco, CA).
4
Hsp90α Regulates CXCL8 Secretion
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Human LECs were cultured in complete medium with 10% FBS until the confluence reached 80%. The culture medium was then replaced with fresh medium containing 1% FBS, in which recombinant Hsp90α or control BSA was administrated. After 16 h, the supernatants were harvested from each culture and stored at −80 °C for further detection. The concentrations of CXCL8 were quantified using the human CXCL8/IL-8 ELISA Kit (D8000C, R&D Systems, Minneapolis, MN, USA).
5
Neutrophil IL-8 production upon stimulation
6
Inflammation-Induced Medium and PHMB-Containing Emulsion
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Two types of conditional media were prepared prior to the experiment. First, inflammation-induced medium (IIM) was prepared by mixing cell culture medium and TSB, which was used for S. aureus cultivation for 24 h and purified by centrifugation and filtration, at 1:200 (v/v). PEENPPCH containing 25 mg/mL PFOB, 60 µg/mL EGF, and 2000 ppm PHMB was immersed in the IIM at 37 °C for 48 h, and then the supernatant (i.e., PEENPPCH-treated IIM; P-IIM) was collected, centrifuged, and stored at 4 °C until use. Afterward, the KERTr cells were separately treated with normal media (control), IIM, P-IIM, or IIM + N-acetyl-D-glucosamine (NADGA; 10 mM) at 37 °C for 12 h and then cellular interleukin-8 (IL-8) expression was analyzed using the human IL-8/CXCL8 ELISA kit (R&D System, Inc., Minneapolis, MN, USA).
7
Acetate-Induced IL-8 Release in Neutrophils
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The release of IL-8 from neutrophils was measured with a human IL-8/CXCL8 ELISA Kit (R&D). Primary neutrophils were stimulated with 1 or 10 mM acetate 30 min prior to incubation with the indicated secondary stimuli for a further 4.5 h. Stimuli were used at the following concentrations: PSMα3 500 nM; fMLF 500 nM; P2Cysk4 200 ng/ml; LPS 100 nM; opsonized USA200 bacteria at MOI of 1. Human IL-8 detection in the cellular supernatant was performed according to the IL-8 ELISA vendor’s manual. Cytokines in mouse serum were detected using the Bioplex Mouse Cytokine Assay (BioRad) and BioRad Multiplex Instrument.
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