Assessment of cytokine and chemokine release of IL-6 and IL-8 was performed using the CytoSet™ sandwich ELISA according to the manufacturer's instructions (Life Technologies; Mulgrave, Vic, Australia). The release of MCP-1, sICAM-1, and sVCAM-1 was performed by sandwich ELISA from R&D Systems (Minneapolis, MN, USA) according to the manufacturer's instructions. The release of PGF2α into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit according to the manufacturer's specifications (Cayman Chemical Company; Ann Arbor, MI, USA). The interassay and intraassay coefficients of variation for all assays were less than 10%.
Competitive enzyme immunoassay kit
Manufactured by Cayman Chemical
Sourced in United States
The Competitive Enzyme Immunoassay (EIA) kit is a laboratory tool used for the quantitative detection and measurement of target analytes in a sample. It employs a competitive binding principle, where the target analyte in the sample competes with a fixed amount of enzyme-labeled analyte for a limited number of antibody binding sites. The resulting signal is inversely proportional to the concentration of the target analyte in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Sandwich elisa, by R&D Systems (5 mentions)
Elisa development kit, by R&D Systems (4 mentions)
Cytoset, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Fujifilm (1 mentions)
Trichloroacetic acid, by Fujifilm (1 mentions)
Micro smash ms 100, by TOMY (1 mentions)
Sandwich elisa, by Thermo Fisher Scientific (1 mentions)
Hplc system, by Thermo Fisher Scientific (1 mentions)
Sandwich elisa, by Merck Group (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Cxcl8, by Thermo Fisher Scientific (1 mentions)
Cxcl5, by R&D Systems (1 mentions)
Il 1a, by R&D Systems (1 mentions)
Il 1b, by R&D Systems (1 mentions)
Cxcl1, by R&D Systems (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Sandwich elisa kit, by Merck Group (1 mentions)
Cytoset sandwich elisa, by Thermo Fisher Scientific (1 mentions)
24 protocols using competitive enzyme immunoassay kit
1
Cytokine and Chemokine ELISA Assay
2
Balanophonin Inhibits Inflammatory Mediators
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BV2 cells were seeded in 6-well plates at a density of 4×105 cells/well and incubated for 24 h. Thereafter, cells were pretreated with balanophonin at concentrations of 1 to 10 μM for 30 min before stimulation with LPS (100 ng/mL) for 24 h. Culture medium was used to measure the inhibitory effect of Balanophonin on the production of PGE2, TNF-α and IL-1β. PGE2 was measured using a competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA). TNF-α and IL-1β were measured using an ELISA development kit (R&D Systems).
3
Measurement of Intracellular cGMP Levels
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Intracellular cGMP concentrations were measured in cells and rat lung tissues as described previously with some modifications.11 (link) In the in vitro assay using SeV, SeV-infected cells were serum-starved for 6 hours or not starved on the day of the cGMP assay and were then incubated for 10 minutes with 1 mmol/l 3-isobutyl-1-methylxanthine (Wako, Tokyo, Japan) in order to inhibit the degradation of cGMP by PDE. Cells were subsequently incubated with 1 × 10−6 M CNP-22 (Bachem AG, Bubendorf, Switzerland) or vehicle (water) for another 10 minutes. The reaction was terminated with 100 µl of 0.1 mol/l HCl.
In the in vitro assay using sildenafil (5 µmol/l) or riociguat (100 µmol/l), on the day of cGMP assay (24 hours after drug stimulation), drug-treated cells were incubated for 20 minutes with 1 mmol/l 3-isobutyl-1-methylxanthine in order to inhibit the degradation of cGMP by PDE. The reaction was terminated with 100 µl of 0.1 mol/l HCl.
In the in vivo assay, fresh rat lungs were placed in 5% trichloroacetic acid (Wako), fractured by MicroSmash MS-100 (TOMY, Tokyo, Japan), and then centrifuged at 15,000 rpm for 10 minutes.
All cGMP concentrations were measured using the competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. cGMP concentrations in lung tissues were normalized by the protein concentrations of the samples.
In the in vitro assay using sildenafil (5 µmol/l) or riociguat (100 µmol/l), on the day of cGMP assay (24 hours after drug stimulation), drug-treated cells were incubated for 20 minutes with 1 mmol/l 3-isobutyl-1-methylxanthine in order to inhibit the degradation of cGMP by PDE. The reaction was terminated with 100 µl of 0.1 mol/l HCl.
In the in vivo assay, fresh rat lungs were placed in 5% trichloroacetic acid (Wako), fractured by MicroSmash MS-100 (TOMY, Tokyo, Japan), and then centrifuged at 15,000 rpm for 10 minutes.
All cGMP concentrations were measured using the competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. cGMP concentrations in lung tissues were normalized by the protein concentrations of the samples.
4
Measuring Pro-Inflammatory Cytokines in BV2 Cells
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For measuring pro-inflammatory cytokines, we seeded BV2 cells in a 6-well plate at a density of 1.5 × 106 cells per well and incubated for 24 h. Cells were pre-treated with A. holophylla soluble fractions and its subfraction and exposed with or without LPS and incubated for the next 24 h. Conditioned medium from the treated cells was collected and stored at −80 °C until further use (this medium can be stored for up to a few months for ELISA). ELISA for TNF-α, IL-1β, and IL-6 was performed using ELISA development kits of the particular cytokine, while PGE2 was measured using a competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA). All experiments were performed as per the protocol provided with the respective ELISA kit.
5
Quantification of cAMP in Preadipocytes
6
Plasma 8-isoprostane Quantification
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Aliquots of plasma (250 μL) samples stabilized with DETPA and BHT were used for the 8-isoprostane (15(s)-8-iso-PGF2α)analysis. Plasma samples were purified initially using C18 cartridges and were analysed using a competitive enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, Michigan, USA) following a procedure reported earlier [34 (link)].
7
Measurement of Inflammatory Mediators
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The levels of IL6 and CXCL8 in the incubation media were measured using sandwich ELISA from Invitrogen per the manufacturer's instructions. The levels of CCL2, CXCL1, and sICAM1 in the incubation media were measured by sandwich ELISA from R&D Systems (Minneapolis, MN, USA) per the manufacturer's instructions. The release of PGF2α into the incubation medium was assayed using a commercially available competitive enzyme immunoassay kit per the manufacturer's specifications (Cayman Chemical Company; Ann Arbor, MI, USA). Interassay and intra-assay coefficients of variation for all assays were less than 10%.
8
Evaluating Inflammatory Responses in BV2 Microglial Cells
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BV2 microglial cells were treated with the samples (NR, RR, and R) and activated with LPS to measure TNF-α, IL-1β, PGE2, and IL-6 levels under neuroinflammatory conditions. BV2 cells were seeded at a density of 1.5 × 106 cells/well in DMEM and incubated for 24 h. The seeded cells were treated with the samples, followed by LPS treatment for microglial activation, and incubated for 24 h. After 24 h of incubation, conditioned medium from the treated plate was collected and used for measuring the levels of PGE2, TNF-α, IL-1β, and IL-6. The collected conditioned medium can be stored at −20°C until later use. PGE2 was measured using a competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA). TNF-α, IL-1β, and IL-6 levels were measured using ELISA development kits (R&D Systems, Minneapolis, MN, USA).
9
Measurement of Plasma 8-Isoprostane Levels
10
Evaluating Inflammatory Mediators in BV2 Cells
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CM from the treated cells was used to evaluate the levels of secreted inflammatory mediators and proinflammatory cytokines, such as PGE2, TNF-α, IL-1β, and IL-6. For this purpose, BV2 cells were seeded in a 6-well or a 24-well plate and treated, as described previously. The treated and LPS-activated cells were incubated for 24 h, whereupon the CM was collected. The cell viability assay was performed on the attached cells, whereas competitive ELISA was used to measure the levels of PGE2, TNF-α, IL-1β, and IL-6 secreted into the CM. ELISA development kits (R&D Systems) were used to measure TNF-α, IL-1β, and IL-6 secretion, whereas a competitive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA) was used to measure PGE2, according to the manufacturers’ instructions. IL-10 and IL-4 secretion was measured using the Mouse Th1/Th2 Uncoated ELISA Kit (Invitrogen).
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