TME-stimulated and vehicle-treated MCF-7 and T47D cells were grown (as described above) for different time points (as described in Figure legends). Then, conditioned media (CM) were removed and CXCL8, IL-6, IFNβ or IFNγ levels were determined by ELISA using standard curves at the linear range of absorbance. To this end, recombinant human CXCL8 (rhCXCL8; #200-08; PeproTech) or rhIFNβ (#300-O2BC; Peprotech) were used. Coating polyclonal antibodies (CXCL8: #500-P28, IFNβ: #500-P32B; PeproTech) and detecting biotinylated rabbit polyclonal antibodies (CXCL8: #500-P28Bt, IFNβ: #500-P32BBT; PeproTech) were used. After the addition of streptavidin-horseradish peroxidase (#016-030-084; Jackson Immunoresearch Laboratories, West Grove, PA, USA), substrate TMB/E solution (#ES001; Millipore, Temecula, CA, USA) was added. For IL-6 and IFNγ, the following ELISA kits were used: IL-6 (#900-TM16; Peprotech), IFNγ (#900-TM27, Peprotech). The reaction was stopped by the addition of 0.18 M H2SO4 and was measured at 450 nm.
Cxcl8
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
CXCL8 is a lab equipment product manufactured by Thermo Fisher Scientific. It is a protein that plays a role in the immune response. The product allows for the detection and quantification of CXCL8 in various samples.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by Thermo Fisher Scientific (5 mentions)
Tnf α, by Thermo Fisher Scientific (4 mentions)
Cxcl5, by R&D Systems (3 mentions)
Cxcl12, by Thermo Fisher Scientific (3 mentions)
Gm csf, by Thermo Fisher Scientific (3 mentions)
Cxcl1, by Thermo Fisher Scientific (3 mentions)
Ccl20, by Thermo Fisher Scientific (3 mentions)
Cxcl10, by R&D Systems (2 mentions)
Rhcxcl 8, by Thermo Fisher Scientific (2 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Elisa kits for ccl2, by Thermo Fisher Scientific (2 mentions)
Ccl11, by Thermo Fisher Scientific (2 mentions)
Ccl17, by Thermo Fisher Scientific (2 mentions)
Cxcl11, by Thermo Fisher Scientific (2 mentions)
Cxcl5, by Thermo Fisher Scientific (2 mentions)
Cxcl2, by Thermo Fisher Scientific (2 mentions)
Il 12p70, by Thermo Fisher Scientific (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Cx3cl1, by R&D Systems (1 mentions)
Matrigel, by BD (1 mentions)
33 protocols using cxcl8
1
Quantifying Cytokine Secretion in Breast Cancer Cells
2
Isolating and Stimulating Human Monocytes
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Whole blood was obtained from healthy volunteers and peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll-Hypaque density gradient centrifugation. CD14+ monocytes were magnetically labeled with CD14 MicroBeads (Miltenyi Biotec, Auburn, CA) and purified on a magnetic-activated cell sorting (MACS) separator following the manufacturer’s protocol. The eluted fraction containing the CD14+ cells were cultured at 5–6 x 106 cells per well in 6-well tissue culture plates using complete medium (RPMI 1640, 10% fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin) supplemented with GM-CSF (50 ng/mL) and IL-4 (50 ng/mL). After incubating for 5 days at 37°C, the cells were washed, counted, and plated in 96-well U-bottom tissue culture plates at 6 x 104 cells per well, and incubated with formulations diluted to 10 μg/mL and 1 μg/mL of GLA, in duplicate. After incubating at 37°C for 24 hours, supernatants were assayed for TNFα, IL-6, IL-12p70, CCL2, CCL5, CXCL8 (eBioscience), CCL4, CXCL5, and CXCL10 (R&D systems). Duplicate wells for each donor and condition were averaged for a single value.
3
Neutrophil Cytokine Secretion Assay
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Neutrophils isolated and stimulated with IL-18 overnight for 14 h and then cell-free supernatants were collected. We then performed quantitative measurements of CXCL8 (Ebioscience, San Diego, California) and CXCL6 (Biolegend, San Diego, California) levels by enzyme-linked immunosorbent assay according to manufacturer's instructions.
4
Cytokine profiling of adjuvant formulations
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Adjuvant formulations were diluted in irrigation-grade saline and added to 96-well U-bottom tissue culture plates. Heparinized whole blood, obtained from healthy donors, was added in duplicate to each formulation, resulting in final GLA concentrations of 10μg/mL or 1μg/mL. Plasma supernatants were harvested after 24 hours of culture at 37°C then assayed for Tumor necrosis factor-alpha (TNFα), Interleukin-6 (IL-6), Interleukin-1-beta (IL-1β), Interferon-gamma (IFN-γ), C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 8 (CXCL8) (eBioscience, San Diego, CA), C-C motif chemokine ligand 4 (CCL4), C-X-C motif chemokine ligand 5 (CXCL5) and C-X-C motif chemokine ligand 10 (CXCL10) (R&D Systems, Minneapolis, MN). Duplicate wells for each donor and condition were averaged for a single value.
5
Pancreatic Cancer Cell Invasion and Migration Assay
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Cells were starved by serum free medium for 24 h prior to invasion and migration assays. Cell invasion and migration assays were both evaluated by the Boyden chamber method using the filter inserts (pore size, 8 μm), pre-coated (for invasion assay) or not pre-coated (for migration assay) with Matrigel in 24-well plates (BD Biosciences, Franklin Lakes, NJ, USA). Each pancreatic cancer cell line (7.5 × 104 cells/insert for MIA PaCa-2, 2 × 105 cells/insert for AsPC-1, or 4 × 104 cells/insert for PANC-1) was seeded on the top chamber. The top chamber was filled with serum-free medium and the bottom chamber was filled with 10% FBS medium. The recombinant chemokines were then set in the bottom chamber at a final concentration of 25 ng/ml for CXCL8 (PeproTech, Rocky Hill, NJ, USA), and 0.5 μM for PGE2, 5 ng/mL for CX3CL1 (R&D Systems, Minneapolis, MN, USA) and C5a (ProSpec-Tany Technology Ltd, Ness Ziona, Israel). After incubation for 8 h in the case of PANC-1, or 48 h in the case of MIA PaCa-2 and AsPC-1cells, cells that passed through the filter and were attached to the lower surface of the filter were counted by staining with 0.01% crystal violet in 25% methanol. Cells attached to the lower surface of the filter membrane were then quantified by cell counting in five non-overlapping fields at × 100 magnification.
6
ELISA Analysis of Cytokines in TB
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The concentrations of GM-CSF, CXCL8 (PeproTech), VEGF, and OSM (R&D Systems) in supernatants of uninfected and M. tuberculosis-infected ΜΦ and in human serum were determined in duplicate by specific sandwich ELISA as described by the manufacturers. Minimum detection limits were 32 pg/ml (GM-CSF), 16 pg/ml (CXCL8), 15.6 pg/ml (VEGF), and 31.25 pg/ml (OSM).
7
Neutrophil Adhesion Assay on E-Selectin, ICAM-1, and CXCL8
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Ibidi flow chambers (0.5 μm Slide VI0.1) were coated with E selectin (rhCD62E-Fc chimera, 5 μg/mL; R&D Systems), ICAM-1 (rhICAM-1, 4 μg/mL; R&D Systems), and CXCL8 (rhCXCL-8, 10 μg/mL, Peprotech) overnight at 4°C. The next day, chambers were blocked with 5% casein. Prior to the start of experiments, isolated human adult neutrophils were incubated for 2 hours with either HBSS (control) or adult or fetal serum, washed twice, and then diluted in HBSS to 1 × 106 cells/mL. Perfusion through the flow chamber was conducted with a high-precision pump (Harvard Apparatus) at a shear stress level of 1 dyne/cm2. Experiments were conducted on a ZEISS AXIOVERT 200 microscope, provided with a ZEISS LD Plan-neofluor objective (20×, 0.4 NA: and a SPOT RT ST Camera). MetaMorph software was used to generate movies for later analysis using Fiji software.
8
Heparinase III-Mediated Glycocalyx Degradation
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F. heparinum heparinase III (Aglyco, Beijing, China) selectively cleaves HS of the glycocalyx (Zeng et al., 2012a (link)). It was demonstrated that HS was dramatically degraded by 15 mU/ml heparinase III in ECs, including HUVECs (Giantsos-Adams et al., 2013 (link)). Cells were treated with 15 mU/ml heparinase III and/or 100 ng/ml CXCL8 (Pepro-Tech, NJ, USA) in basic HUVEC culture medium (Allcells, Shanghai, China) with 1% bovine serum albumin (BSA) for indicated times, respectively.
9
Fluorescent Calcium Flux Assay for GPCR
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U87.CD4 cells that stably express either human CCR7, CXCR2, CCR5 or CXCR4 were seeded (20,000 cells/well) in gelatin-coated black-walled polystyrene 96-well plates with clear bottom and incubated overnight at 37°C and 5% CO2. The next day, a fluorescent Ca2+-sensitive dye solution (Fluo-2 AM) was prepared as described before (Claes et al., 2018 (link)). Culture medium was removed, and cells were incubated for 45 min at room temperature in the dark. Meanwhile, 96-well polypropylene plates containing 5-fold concentrated compound dilutions and 5-fold concentrated solution of chemokine ligands (CCL19, CXCL8, LD78-β, CXCL12, respectively; all purchased from PeproTech) were prepared for use with the FLIPR Tetra device (Molecular Devices) as described before (Claes et al., 2018 (link)). The antagonistic properties of the compounds were calculated based on their capacity to inhibit the Ca2+ release induced by a fixed concentration of chemokine (i.e. 50 ng/mL final concentration for CCL19, CXCL8 and CXCL12 and 100 ng/ml for LD78-β), as described (Claes et al., 2018 (link)). Exactly the same protocol was used to record calcium responses in Chinese hamster ovary (CHO)-K1 cells, upon stimulation with adenosine triphosphate (ATP, purchased from Sigma).
10
Modulation of Cell Signaling Pathways
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Recombinant human LIF (#300-05, 20 ng/mL), CXCL3 (#300-40, 20 ng/mL) and CXCL8 (#200-08M, 20 ng/mL) were all purchased from Peprotech. LIF neutralizing antibody (AB-250-NA, 2 μg/mL) was purchased from R&D, and CXCL3 neutralizing antibody (PP1014P2, 5 μg/mL) was purchased from Origene. Stattic (#HY-13818), LY3214996 (#HY-101494), PD98059 (#HY-12028), SB 203580 (#HY-10256), LY294002 (#HY-10108), JSH-23 (#HY-13982), BAY 11-7082 (#HY-13453), T-5224 (#HY-12270), TK216 (#HY-122903), TAT-DEF-Elk-1 (#HY-P2262A), SB225002 (#HY-16711), Reparixin (#HY-15251), and EC330 (#HY-100949) were all purchased from Med Chem Express. SB-505124 (#M2250) was purchased from Abmole. A list of the concentrations and targets used for the inhibitors is provided in Supplementary Table 1 .
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