At the end of treatments, cell culture supernatants were recovered and centrifuged at 12,000 rpm for 6 min at 4 °C to remove debris and floating cells. The resulting supernatants were collected and stored at −20°C until the test was performed. IL-6 and IL-8 protein levels were measured by sandwich ELISA, according to the manufacturer guidelines (Life Technologies, Monza, Italy). The absorbance of each sample, whose color intensity is proportional to the amount of cytokine, was measured at 450 nm and 630 nm. The amount of proteins in pg/mL was calculated based on a standard curve.
Sandwich elisa
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Austria
Sandwich ELISA is a type of enzyme-linked immunosorbent assay (ELISA) used to detect and quantify the presence of a specific protein in a sample. It involves the use of two antibodies: one that is immobilized on a solid surface to capture the target protein, and another that is labeled with an enzyme and used to detect the captured protein.
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Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Sandwich elisa, by R&D Systems (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
96 well plate, by Greiner (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Multiplex luminometry, by Bio-Rad (1 mentions)
Luminex 200 platform, by Bio-Rad (1 mentions)
Solid phase sandwich elisa, by R&D Systems (1 mentions)
Synergy ht microplate reader, by Agilent Technologies (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Sunrise spectrophotometer, by Tecan (1 mentions)
Cytometric bead array multiplex assay, by BD (1 mentions)
Magnetic beads, by STEMCELL (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Elisa plate reader, by Agilent Technologies (1 mentions)
Competitive enzyme immunoassay kit, by Cayman Chemical (1 mentions)
Benchmark plus microplate reader, by Bio-Rad (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
60 protocols using sandwich elisa
1
Quantification of Secreted Cytokines
2
Quantification of Inflammatory Markers
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The release of MCP-1, TNF-α, IL-6 and IL-8 was performed by sandwich ELISA according to the manufacturer's instructions (Life Technologies; Mulgrave, Vic, Australia). The concentration of IL-1β, sICAM-1 and sVCAM-1 in the media was performed by sandwich ELISA according to the manufacturer's instructions (R&D Systems; Minneapolis, MN, USA). All data were corrected for total protein and expressed as either pg or ng per mg protein. The protein content of tissue homogenates was determined using BCA protein assay (Thermo Fisher Scientific; Scoresby, Vic, Australia), using BSA as a reference standard, as previously described [15] (link). The calculated interassay and intraassay coefficients of variation (CV) were all less than 10%.
3
Multiplex Analysis of Inflammatory Markers
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All measurements were made in duplicate using commercially available kits and manufacturers guidelines. Serum IL-1β, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17, GMC-SF, TNFα and MCP-1 were simultaneously measured by multiplex luminometry (Bio-Rad, Hemel Hempstead, UK). Samples were analysed using a Bio-Plex Luminex200 platform equipped with a 635 nm red and 532 nm green laser using Bio-Plex Manager™ software. Detection of C-reactive protein (CRP) was by high sensitivity ELISA using a commercial kit (IBL International, Hamburg, Germany). Plasma adiponectin and leptin were assessed separately by solid phase sandwich ELISA (R&D Systems, Abingdon, UK). Plasma insulin levels were determined by sandwich ELISA (Life Technologies, Paisley, UK). Plasma glucose, cholesterol and triglycerides (Instrumentation Laboratories, Warrington, UK) were determined photometrically on an automated chemistry analyser (iLab 650, Instrumentation Laboratories, Warrington, UK).
4
Quantification of IL-6 in HMDM Supernatant
5
Assessing IL-31 in Bet v 1-Specific T-Cell Clones
6
Quantifying IL-22 in Cell Lines
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IL-22 in U87MG and U118MG cell line supernatants was detected by sandwich ELISA (Peprotech), according to manufacturer’s instructions. Concentrations below the detection limit (5pg/mL) were considered undetectable.
7
Quantification of Nitric Oxide and Cytokine Release
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Quantification of NO accumulation in cell media was performed using the Griess Reagent (Sigma–Aldrich®—Poole, Dorset, UK), as described before [10 (link)]. For analysis, 100 µL of cell culture medium of each condition were used and the manufacturer’s instructions followed. The absorbance (Abs) was read at 540 nm in a Synergy HT microplate reader (Biotek). For ELISA, cell media was collected from the plate and immediately frozen at −80 °C until analysis. TNF-α release was assayed by sandwich ELISA according to the manufacturer’s instructions (PeproTech®, Princeton Business Park, Rocky Hill, NJ, USA) as previously performed [10 (link)]. The Abs405 was measured using a Synergy HT microplate reader (Biotek) at room temperature (RT). Adherent cells were lysed with RIPA for protein quantification and Griess/ELISA results were normalized for total protein.
8
Quantification of Astrocyte-Secreted Cytokines
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IL-1β and TNF-α were determined in the astrocyte CM. Samples were centrifuged at 14.000 g for 40 min. Supernatants were collected and protein content was assayed by the BCA method. IL-1β and TNF-α levels were determined by sandwich ELISA, according to the manufacturer’s protocol (eBioscience, San Diego, CA, USA). For the assay, 100 µl of samples were added per ELISA plate well and incubated at 4 °C overnight. A calibration curve with recombinant cytokine was included. Detection antibody was incubated at room temperature for 1 h and the reaction developed with avidin–HRP and substrate solution. Absorbance was measured at 450 nm with reference to 570 nm with the microplate reader Synergy HT (Biotek Instruments).
9
Spleen cell response to peanut allergens
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Spleen single cell suspensions (2.5×106/ml) were incubated in the presence or absence of PE, Ara h 1, 2, 3 or 6 (100 μg/ml) for 96 h at 37°C in complete RPMI1640 with 10% FCS. Culture supernatants were harvested and stored at −20°C until analysis. Levels of IL-5 and IL-13 in culture supernatants were determined by sandwich ELISA according to the instructions of the manufacturers (eBioscience, Austria). The detection limit is 5 pg/ml for both ELISA’s.
10
Cytokine Profiling in Cell Cultures
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The concentrations of IL-1β (from 18 h culture supernatants), and IL-17A and IFN-γ (from 72 h culture supernatants), were determined using sandwich ELISA (eBioscience, USA), following the manufacturers’ instructions. The concentrations of IL-6, IL-23, IL-12p70, IL-10 and TFN-α (from 18 h culture supernatants) were determined using a multiplex electrochemiluminescence assay (V-plex, Meso Scale Discovery, USA).
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