PTC, MPV, and PDW were measured by using a multi-function automated blood cell analyzer (XT-4000i, Sysmex, Japan). Levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 in the plasma were measured by using enzyme-linked immunosorbent assay (ELISA) techniques (R&D Systems, Minneapolis, MN, USA). All procedures followed standard protocols (included in the ELISA kits). Spectrophotometry was used to detect the intensity of transmitted light. Data were expressed as ng/mL.
Elisa technique
Manufactured by R&D Systems
Sourced in United States, United Kingdom
ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used analytical technique for the detection and quantification of specific proteins, antibodies, hormones, or other molecules in a sample. The core function of ELISA is to utilize antibodies or antigens immobilized on a solid surface to capture and detect the target analyte in the sample, with the aid of an enzyme-linked detection system.
Automatically generated - may contain errors
Lab products found in correlation
Vacutainer plus serum blood collection tubes, by BD (2 mentions)
Architect i1000sr, by Abbott (2 mentions)
Particle enhanced immunoturbidimetric assay, by Abbott (2 mentions)
Architect ci8200, by Roche (2 mentions)
Xt 4000i, by Sysmex (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Rpmi 1640 with l glutamine, by Merck Group (1 mentions)
A 400, by Olympus (1 mentions)
Immulite 2000, by Siemens (1 mentions)
Ysi model 2300 stat plus, by Yellow Springs Instruments (1 mentions)
Elisa technique, by ALPCO (1 mentions)
19 protocols using elisa technique
1
Hematological and Inflammatory Biomarkers
2
Liver Oxidative Stress Biomarkers
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The liver portions were homogenized in a cold physiological buffer of pH 7.4 (1:10, w/v), and the total protein concentrations in liver samples were measured according to a bicinchoninic acid (BCA) test (Santa Cruz, CA, USA). Thiobarbituric acid reactive substance (TBARS) and GSH levels were measured, using ELISA kits (Cayman Chemical Co., Ann Arbor, MI, USA). The hepatic levels of TNF-α, IL-6, and MDA were determined by following the ELISA techniques (R&D Systems Inc., Minneapolis, MN, USA). In post-mitochondria supernatants of liver samples, the enzymatic activities of CAT and GPX were measured using ELISA kits (R&D Systems Inc., Minneapolis, MN, USA).
3
Lung Tissue Explant Culture for Cytokine Analysis
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The use of lung tissues resected from patients was approved by the regional investigational review board (CPP Ile de France VIII, France) and written informed consent was obtained from each patient. Human lung parenchyma was obtained from 14 patients (age: 66 ± 3 years; males/females, 7/7; non-smokers: 2; smokers and ex-smokers: 8 and 4; pack years: 44 ± 6) undergoing surgical resection for lung carcinoma and who had not received recent chemo/steroid therapy. The procedure for preparation of lung explants has been described elsewhere (Hackett et al., 2008 (link); Buenestado et al., 2013 (link)). Explants (5 fragments of 3–5 mm3, total weight ∼50–100 mg) were maintained overnight at 4°C in RPMI supplemented with 2 mM L-glutamine, 100 μg·mL−1 streptomycin and 100 U·mL−1 penicillin. The fragments were distributed into six-well plates and incubated with olodaterol or vehicle (DMSO) for 1 h before LPS stimulation (1 μg·mL−1). The maximal DMSO concentration applied to explants in culture did not exceed 0.01% and did not alter the production of cytokines or TNF-α. After 24 h, supernatants were collected and stored at −80°C. Production of cytokines was measured in medium supernatants with elisa techniques (R&D Systems, Minneapolis, MN, USA).
4
Synergistic Effects of IL-17, TNF-α, and IL-1β on IL-6 Production
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The synergistic effect of IL-17 (50 ng/ml) and TNF-α (500 pg/ml) on the production of IL-6 by synoviocytes was already evaluated in previous papers [11 (link),12 (link)]. Here a dose-response curve was established, comparing the known effect of IL-17/TNF combination to the one of IL-17 in association with IL-1β (10 and 100 pg/ml). IL-1β was first used alone or in association with IL-17 to verify the synergistic effect.
IL-6 production was quantified in synoviocyte and co-culture supernatants of day 5 by standard ELISA techniques (R&D system, San Diego, CA, USA). IL-1β and TNF-α production was measured in PBMC and monocyte cultures or in co-cultures in the presence or not of Cd. Absorbance at 450 nm was measured using the VICTOR X4 plate reader and results were obtained by subtracting the background read at 540 nm and compared to the regression curve obtained by standards of R&D system kit following manufacturer instructions.
IL-6 production was quantified in synoviocyte and co-culture supernatants of day 5 by standard ELISA techniques (R&D system, San Diego, CA, USA). IL-1β and TNF-α production was measured in PBMC and monocyte cultures or in co-cultures in the presence or not of Cd. Absorbance at 450 nm was measured using the VICTOR X4 plate reader and results were obtained by subtracting the background read at 540 nm and compared to the regression curve obtained by standards of R&D system kit following manufacturer instructions.
5
Plasma Biomarker Quantification Protocol
6
Cytokine Production in Activated PBMCs
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Peripheral blood mononuclear cells were isolated from venous blood by density gradient centrifugation method. Isolated cells (2 × 106) were transferred to 6-well cell culture plates and suspended in 2 ml of standard cell culture medium (RPMI-1640 with L-glutamine; Sigma Aldrich; Germany) supplemented with 10% heat-inactivated foetal bovine serum (FBS, PAA; Austria). Culture plates were placed at 37°C in an incubator providing a humidified atmosphere containing 5% CO2 for 1 hour. After preincubation the cells were stimulated with lipopolysaccharide (LPS) form E. coli (Sigma Aldrich; Germany) in a concentration of 1 µg/ml/106 cells. As a control, cells were incubated with 0.9% NaCl. After four hours of incubation, cell suspensions were collected and transferred into sterile 2-ml microcentrifuge tubes. Samples were centrifuged for 10 minutes at 1500 × g and 23°C. Supernatants were collected and stored at –40°C until analysed. The cell supernatants were assayed for the presence of IL-1β, IL-4, and IL-6 using ELISA technique (R&D Systems, USA).
7
Serum IL-6 and PON1 Activity Assay
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Blood samples were taken from the antecubital vein at 11 a.m. on an empty stomach and were collected in BD Vacutainer Plus serum blood collection tubes (ref. 367815). The samples were then kept at room temperature for 30 min in order to coagulate. The coagulated part was then separated by centrifuging the samples at 4000 rounds/min for 10 min in a refrigerated centrifuge. After centrifuging, the supernatant liquid was transferred to 0.5 mL aliquots that were frozen and stored at −80 °C. Finally, once the samples thawed, the concentration of IL-6 in serum was determined by means of the ELISA technique (R&D Systems). PON1 activity was determined by using 4-nitrophenyl acetate with an automatic clinical biochemistry analyser (Olympus A 400, Tokyo, Japan) [29 (link)].
8
Cytokine Profiling of T Cell Activation
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In order to measure the systemic effects of T cell activity and inflammation, we measured the levels of serum IL17, IL4, TNFα, IFNγ and ICOSL by the ELISA technique (R&D Systems®, Minneapolis, MN, USA). The production of cytokines by PBMCs stimulated with B7.1-Fc or B7h-Fc was evaluated by the ELISA technique in cellular supernatants. The levels of IL17, IL4, TNFα and IFNγ (R&D Systems®, Minneapolis, MN, USA) were measured. All the experiments were performed in duplicate.
9
Cytokine and Chemokine Quantification
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CXCL1/KC, MPO, IL-6, and TNF-α concentrations were determined in BAL fluid using a standardized ELISA technique (R&D Systems, Minneapolis, MN). Recombinant murine proteins were used to generate standard curves.
10
Plasma Hormone Measurements in Metabolic Studies
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Plasma-soluble leptin receptor concentrations were measured by an ELISA technique (R&D Systems, Minneapolis, MN, USA) with a sensitivity of 0.06 ng ml−1. Plasma leptin, plasma PYY3–36 and plasma ghrelin were measured by radioimmunoassay (Millipore, Billerica, MA, USA). All quality controls (prepared by the manufacturer) were within acceptable limits. Radioimmunological determinations of total plasma GLP-1 were performed as described.26 (link), 27 (link), 28 (link) The analytical detection limit was 1 pmol l−1 and intra- and inter-assay coefficients of variation were <6% and <15%, respectively. Plasma glucose was measured with the glucose oxidase technique (YSI model 2300 STAT Plus; Yellow Springs Instruments, Yellow Springs, OH, USA). Serum insulin concentrations were measured using Immulite 2000 solid-phase chemiluminescent immunometric assays (Immulite 2000; Siemens, Erlangen, Germany). Fat percentage was assessed using dual-energy X-ray absorptiometry scanning (Hologic discovery A, Bedford, MA, USA). Blood pressure was measured using semi-automatic upper arm blood pressure monitor (Boso Mercurius E; Bosch+Sohn, Jungingen, Germany).
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