HGK were seeded on 12-well plates (5 × 105 cells/well) and incubated overnight. Treatments are described in the figure legends. The amounts of IL-6, IL-8, and IFN-β in media were assessed using the OptEIA Human IL-6 ELISA set (555220; BD Biosciences, San Diego, CA, USA), OptEIA Human IL-8 ELISA set (555244; BD Biosciences), and Human IFN-beta Quantikine ELISA (DIFNB0; R&D Systems), respectively.
Opteia human il 8 elisa set
Manufactured by BD
Sourced in United States
The BD OptEIA™ - Human IL-8 ELISA Set is a laboratory equipment product designed for the quantitative measurement of human interleukin-8 (IL-8) in biological samples using the enzyme-linked immunosorbent assay (ELISA) technique.
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Lab products found in correlation
Opteia human il 6 elisa set, by BD (3 mentions)
Cytotox 96 assay, by Promega (1 mentions)
Synergy ht plate reader, by Agilent Technologies (1 mentions)
Immuno maxisorp 96 well plates, by Thermo Fisher Scientific (1 mentions)
Model benchmark, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Penicillin, by InvivoGen (1 mentions)
Streptomycin, by InvivoGen (1 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
L glutamine, by InvivoGen (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Human tnf elisa set, by BD (1 mentions)
Opteia human gm csf elisa set, by BD (1 mentions)
Opteia human il 10 elisa set, by BD (1 mentions)
10 protocols using opteia human il 8 elisa set
1
Cytokine Measurement in HGK Cells
2
Evaluating Aspergillus Infection Biomarkers
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Epithelial cell toxicity was determined by measuring lactate dehydrogenase (LDH) release using the CytoTox 96 assay (Promega, Sydney, NSW, Australia, #G1780). The neutrophil chemotactic cytokine interleukin-8 (IL-8) was measured using the OptEIA Human IL-8 ELISA set (BD, #555244) according to manufacturer’s instructions, and IL-6 was measured using an in-house time-resolved fluorometry detection system [47 (link)]. Aspergillus galactomannan was examined by the Platelia Aspergillus Ag sandwich microplate assay (Bio-Rad, Sydney, NSW, Australia, #62794) with positivity determined by a positive index of >1.5.
3
Quantifying TLR5-Mediated CXCL8 Response
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The read-out for TLR5 activity in all experiments was CXCL8 secretion into cell line culture supernatants. Cell lines were prepared at 5 × 105 cell per ml and 180 μl/well was added to each well in a 96-well plate, with the addition of 20 μl/well flagellin at final concentrations as required (see results). Following overnight incubation, cell culture supernatants were harvested for assays. Human CXCL8 protein levels were measured by ELISA using the BD OptEIA human IL-8 ELISA set (BD Biosciences) following the manufacturer’s instructions. Bovine CXCL8 protein release was measured by ELISA as described previously7 (link). All ELISAs were carried out using Nunc-Immuno Maxisorp 96 well plates, with all samples and standards in triplicate. The plates were read using a Synergy HT plate reader (BioTek).
4
Inflammatory Cytokine Secretion in CSE-Treated Calu-3 Cells
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Calu-3 cells were seeded (27,000 cells/cm2 in 100 µl of DMEM-10% FBS medium) in 96 well-plates and grown to confluence. The cells were then incubated with 100 μg/ml CSE, 100 μg/ml CSE plus 10 μM IKK-2 inhibitor, 100 μg/ml CSE plus 5 mM NAC or the vehicle for 24 h. IL-6 and IL-8 were measured from supernatants using the human IL-6 and IL-8 ELISA set (BD OptEIA™ - Human IL-8 ELISA Set, and BD OptEIA™-Human IL-6 ELISA Set, BD Biosciences, San Diego, CA). Measurements were performed using a microplate reader according to manufacturer´s instructions (model Benchmark, Bio-Rad).
5
Quantifying IL-8 Secretion in Treated Cells
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Cells were seeded in a 12-well plate in RPMI media with 10% FBS and 1% ABAM. At confluency, the cells were serum-starved for 6 h and then treated with DMSO 4 nM equivalent, CBZ 2.5 nM, T BL NPs 2.5 nM, NT CBZ NPs 2.5 nM, and T CBZ NPs 2.5 nM in 1% FBS. Aliquots of the supernatant were taken after 6 h. The concentration of IL-8 was assessed using BD OptEIA, Human IL-8 ELISA set, by BD Biosciences (Cat. # 555244, San Diego, CA, USA) according to the manufacturer’s protocol. The absorbance was read at 450 nm after stopping the reaction with 1 M H3PO4.
6
IL-8 Quantification in Breast Cell Lines
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MCF10A and MCF7 cells seeded in 6-well plates (6.0 × 105 and 4.0 × 104 cells per well respectively) were transfected with 500 ng expression plasmid and 2 μg total DNA. Medium supernatant was collected 48 h post-transfection. Cells were rinsed with phosphate buffered saline (PBS) and lysed in IPLS lysis buffer including Tris–HCl pH 7.5 20 mM, 120 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid, 0.5% NP40, 10% glycerol and protease inhibitor (#11873580001, Roche). IL-8 levels were quantified using the BD OptEIA Human IL-8 ELISA Set (BD Bioscience, East Rutherford, New Jersey, USA) following manufacturer's instructions. Total protein levels were quantified with a Bicinchoninic Acid Assay (BCA) kit (Pierce). Results are presented as means of three biologically independent duplicates. In each experiment, the IL-8 level/protein ratio is calculated as a percentage of the control.
7
Cytokine-Induced IL-8 Secretion in HT29/C1 Cells
8
Interleukin Secretion Quantification
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Cells were seeded in 96 well-plates. Following damaging protocols, the medium was replaced by 50 µl of fresh culture medium with 1% FCS. Interleukins 6 and 8 were measured from frozen 24 h supernatant using the Human IL-6 and IL-8 ELISA sets (BD OptEIA™ - Human IL-8 ELISA Set, and BD OptEIA™-Human IL-6 ELISA Set, BD Biosciences). Measurements were performed using a microplate reader (Benchmark, Bio-Rad). Values were normalized to 10,000 cells.
9
TLR5-mediated IL-8 secretion in HEK293 cells
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HEK 293 cells stably transfected with the murine TLR5 (InvivoGen, Toulouse, France) were cultured in DMEM (Biochrome, Berlin, Germany) containing 10% FCS (Biochrom, Berlin, Germany), L-glutamine (0.15 mg/ml), penicillin (100 U/ml), streptomycin (100 µg/ml), and blasticidin (10 µg/ml) (InvivoGen). Wild-type HEK 293 cells were cultured without blasticidin as indicated above. For TLR5-activation assays, 4 × 104 cells per well were seeded in 48-well plates and cultured in DMEM containing 2% FCS, L-glutamine, penicillin, and streptomycin overnight. Cells were stimulated for 22 h with equimolar amounts rFlaA, rFlaA:Artv1, or rFlaA:Artv1hyp. Subsequently, human IL-8 secreted in the supernatant was quantified by ELISA using the BD OptEIA human IL-8 ELISA Set (BD Biosciences, Heidelberg, Germany).
10
Cytokine Profiling of Immune Cells
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