Paraffin embedded prostate cancer tissue blocks were from the Vancouver Prostate Centre Tissue Bank. Patient consent was reviewed and approved by the University of British Columbia Clinical Research Ethics Board (certificate no. H09-01628). Immunohistochemical staining was conducted as previously described 56 (link) using the Ventana DiscoverXT Autostainer (Ventana Medical System) with enzyme labeled biotin streptavidin system and solvent-resistant DAB Map kit. Antibodies used in IHC include anti-FoxA1 (Abcam; ab23738), anti-SYP (Abcam; ab32127), anti-chromogranin A (Millipore; MAB5268), and anti-IL-8 (R&D systems).
Anti il 8
Manufactured by R&D Systems
Sourced in United States, Germany
Anti-IL-8 is a laboratory reagent used for the detection and quantification of IL-8 (Interleukin-8) in biological samples. IL-8 is a chemokine that plays a role in the inflammatory response. This product is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 6, by R&D Systems (3 mentions)
Ab23738, by Abcam (2 mentions)
Ab32127, by Merck Group (2 mentions)
Ventana discoverxt autostainer, by Roche (2 mentions)
Mab5268, by R&D Systems (2 mentions)
Il 17, by R&D Systems (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Goat anti mouse hrp, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Anti il 17, by R&D Systems (1 mentions)
Anti kim 1, by R&D Systems (1 mentions)
Anti il 23, by R&D Systems (1 mentions)
Anti cd3 and anti cd28, by BD (1 mentions)
Anti il 22, by R&D Systems (1 mentions)
Recombinant human ifn γ, by R&D Systems (1 mentions)
Il 22, by R&D Systems (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Il 23, by R&D Systems (1 mentions)
Anti il1ra, by R&D Systems (1 mentions)
12 protocols using anti il 8
1
Prostate Cancer Tissue Immunohistochemistry
2
Proteolytic Processing of IL-8 by MMPs
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In total, 10 µg/mL of activated MMP, in some cases pretreated with 10 µg/mL SSL1 or SSL5, was incubated with 0.1 µg/mL of IL-8 (77 aa) overnight in activation buffer supplemented with 0.05% human serum albumin. The 72 aa and 77 aa variant of IL-8 were taken along as controls and treated exactly the same as the MMP samples. Samples (10 µL) were loaded on 16.5% Tris-Tricine gels and run for 2 h at 100 V, after which they were transferred to a blotting membrane using Trans-Blot® Turbo™ Transfer System (Bio-Rad). Blots were blocked with 4% skimmed milk in PBS-T. Subsequently, the membrane was incubated with 10 µg/mL anti-IL8 (R & D Systems, clone 6217.111) and goat anti-mouse HRP (1:10,000, Bio-Rad) for 1 h at 37 °C. In between incubation steps, the blots were extensively washed with PBS-T. Finally, blots were developed with ECL (Thermo Fisher Scientific) and visualized on a LAS 4010 imaging system.
3
Quantifying IL-8 Levels via ELISA
4
Prostate Cancer Tissue Immunohistochemistry
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Paraffin embedded prostate cancer tissue blocks were from the Vancouver Prostate Centre Tissue Bank. Patient consent was reviewed and approved by the University of British Columbia Clinical Research Ethics Board (certificate no. H09-01628). Immunohistochemical staining was conducted as previously described 56 (link) using the Ventana DiscoverXT Autostainer (Ventana Medical System) with enzyme labeled biotin streptavidin system and solvent-resistant DAB Map kit. Antibodies used in IHC include anti-FoxA1 (Abcam; ab23738), anti-SYP (Abcam; ab32127), anti-chromogranin A (Millipore; MAB5268), and anti-IL-8 (R&D systems).
5
Wound Healing Assay with Cell Lines
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A549 and H292 cells were seeded in a 6-well plate and were cultured to confluence. Three circular wounds were prepared in each well using a 200-µL pipette tip as previously described [16 (link)]. After washing with PBS to eliminate debris, cells were allowed to recover for one hour and then stimulated with oil, silica, FOS (10 μg/mL), and anti-IL-8 (1 μg/mL) (R&D Systems, Minneapolis, MN, USA), as already described. Images were captured using a digital camera connected to an inverted phase-contrast optical microscope 0, 24, and 48 h after wound creation. The wound area was measured using the ImageJ program in order to assess the remaining wound sizes and wound closure rates.
6
Cytokine Profiling in Immune Cell Activation
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Recombinant human IFN-γ, IL-17, IL-22, IL-23, TNF-α, IL-6 and IL-8 were purchased from R&D Systems (Minneapolis, MN). Anti-human IFN-γ, anti-IL-17, anti- IL-22, anti-IL-23, anti-IL-6, anti-IL-8 and anti-KIM-1 antibodies were purchased from R&D Systems. Anti-CD3 and anti-CD28 were obtained from BD Biosciences (San Diego, CA), and 1,25(OH)2D3 was obtained from Sigma (St. Louis, MO).
7
Endothelial Barrier Disruption by Tumor Factors
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We continuously measured the impedance of an endothelial cell monolayer as previously reported [22 (link)]. HUVECs, at a count of 105, were grown to confluence in standard medium on planar gold-film electrodes deposited on the bottom of an 8-well electrode array (ECIS Cultureware 8W10E+, Applied BioPhysics Inc., Troy, NY, USA). Measurements were performed under standard cell culture conditions. Impedance was continuously measured using an ECIS 1600R instrument (Applied BioPhysics, Inc.) at a sampling frequency of 4 kHz, as this was proven to be the most sensitive frequency in assessing endothelial barrier function [23 (link)]. After 20 h, half of the endothelial cell culture medium was replaced by starvation medium as a control or by tumour cell SN with or without distinct inhibitors ((anti-IL1ra 320 ng/ml (R&D systems, Wiesbaden, Germany); anti-CXCL1 150 ng/ml (LSBio, Seattle, USA); anti-IL6 120 ng/ml (R&D systems, Wiesbaden, Germany); anti-IL8 50 ng/ml (R&D systems, Wiesbaden, Germany).
8
Quantification of IL-8 Levels via ELISA
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ELISA assay was performed by coating 96-well plates with 1 mg per well of anti-IL-8 (R&D Systems). Before the subsequent steps in the assay, the coated plates were washed twice with PBST. All reagents and coated wells used in this assay were incubated for 2 h at room temperature. Following exposure to the medium, the assay plates were exposed sequentially to each of the biotin-conjugated secondary antibodies, as well as AP and ABTS substrate solution containing 30% H2O2. The plates were read at an absorbance of 405 nm. Appropriate specificity controls were included and all samples were run in duplicate.
9
Recombinant MIF and Antagonist Preparation
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Recombinant human MIF (rhMIF) was prepared as described elsewhere (46 (link)) (endotoxin content < 0.1 EU/ml). MIF antagonist MIF098 [3-(3-hydroxybenzyl)-5-methylbenzooxazol-2-one] was dissolved in DMSO at a concentration of 149 µM (47 (link)). The neutralizing anti-MIF monoclonal antibody (clone NIHlllD.9) was obtained from ascites after purification using protein A/G spin column and resuspended at 5.15 mg/ml (48 (link), 49 (link)). A CD74 blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα (MiltenyiBiotec), and the cytokine neutralizing antibodies anti-IL-8 (R&D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα (BioLegend) were obtained.
10
Neutrophil and CD8+ T Cell Migration Assays
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Migration of neutrophils, isolated from PBMC of HDs by magnetic beads (EasyStep enrichment kit, StemCell Technologies), was assessed in transwell plates (5 µm pore size, Corning Costar), towards Th17 supernatants or recombinant cytokines (IL-17, R&D Systems, 50 ng/mL; IL-8, R&D Systems, 100 ng/mL; GM-CSF, R&D Systems, 100 ng/mL), for 90 min at 37°C. In specific experiments, anti-IL-8 or anti-GM-CSF antibodies (10 μg/mL, R&D Systems) were added to Th17 supernatants.
Migration of CD8+ T cells, sorted from PBMC of HDs by magnetic microbeads (Miltenyi Biotec) and pre-activated overnight with anti-CD3/CD28 antibodies, was evaluated in transwell plates (5 µm pore size) towards Th17 supernatants, supernatants of HMEC cells, untreated, or exposed overnight to Th17 supernatants or recombinant proteins (IL-17, 50 ng/mL; TNF-α, 1 ng/mL, R&D Systems; IFN-γ, 1 ng/mL, Biolegend; or their combination), or towards CCL5 (60 and 200 ng/mL, R&D Systems) and/or CCL20 (300 and 1000 ng/mL, R&D Systems). Depletion of CCL5 and/or CCL20 from Th17-derived supernatants was obtained by specific capture antibodies (R&D Systems). Cell migration was quantified by flow cytometry.
Migration of CD8+ T cells, sorted from PBMC of HDs by magnetic microbeads (Miltenyi Biotec) and pre-activated overnight with anti-CD3/CD28 antibodies, was evaluated in transwell plates (5 µm pore size) towards Th17 supernatants, supernatants of HMEC cells, untreated, or exposed overnight to Th17 supernatants or recombinant proteins (IL-17, 50 ng/mL; TNF-α, 1 ng/mL, R&D Systems; IFN-γ, 1 ng/mL, Biolegend; or their combination), or towards CCL5 (60 and 200 ng/mL, R&D Systems) and/or CCL20 (300 and 1000 ng/mL, R&D Systems). Depletion of CCL5 and/or CCL20 from Th17-derived supernatants was obtained by specific capture antibodies (R&D Systems). Cell migration was quantified by flow cytometry.
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