The ex vivo levels of TNF-α, IL-1β, sTNFR-R2, and IL-1RA were measured using multianalyte assay ELLA (R&D systems, Minneapolis, MN, USA) and hsCRP using (Roche/Hitachi cobas c systems). The in vitro evoked-release of TNF-α, IL-1β, IL-10, IL-4, IL-1RA, CCL2, CCL3, and CCL4 were determined using a custom-made U-plex (MSD, Maryland, MD, USA). Supernatants were diluted 100-fold. In case the inflammatory marker level was below the lower limit of quantification (LLOQ) the value was substituted with half the LLOQ value (Croghan and Egeghy, 2003 ). These inflammatory markers were selected as these have been shown to be related to musculoskeletal pain (Barbe and Barr, 2006 (link); Teodorczyk-Injeyan et al., 2011 (link); Sterling et al., 2013 (link); Ethemoǧlu and Erkoç, 2020 (link); Farrell et al., 2020 (link); Zhou et al., 2021 (link)).
U plex
Manufactured by MSD
Sourced in United States
The U-plex is a versatile lab equipment designed for various applications. It offers a core function of providing a uniform and consistent environment for experiments and analyses. The U-plex is engineered to maintain precise temperature, humidity, and other environmental conditions to ensure reliable and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Cobas c system, by Hitachi (1 mentions)
Human il 10 uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Human il 6 duoset elisa kit, by R&D Systems (1 mentions)
Human mouse tgfβ1 uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Duoset ancillary reagent kit 2, by R&D Systems (1 mentions)
4 protocols using u plex
1
Inflammatory Markers in Musculoskeletal Pain
2
Multiplex Assay for Cytokine Profiling
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Supernatants of M2MQs were assayed for cytokines and other released proteins. An electrochemiluminescence multiplex assay (U-Plex, Biomarker Group 1 Assays, Meso Scale Discovery (MSD), Rockville, MD, USA) was used to simultaneously quantify M-CSF, IL-1RA, TARC, IL-6, MCP-1, Eotoxin-2, IL-23, MDC, IL-13 and IL-4. The protocol and data quantification using an MSD 1300 reader was run following the manufacturer’s instructions. Enzyme-linked immunosorbent assays (ELISAs) were used for quantitation of IL-10 using Human IL-10 uncoated ELISA kit and TGFβ1 using Human/Mouse TGFβ1 uncoated ELISA kit (both Invitrogen) in Maxisorp NUNC-Immuno flat bottom 96-well plates (Thermofisher).
Fibroblast supernatants were assayed for Pro-collagen I using DuoSet ELISA Human Pro-Collagen I a1/COLIA1 kit, fibronectin using DuoSet Human Fibronectin and IL-6 using DuoSet Human IL-6 ELISA kit with the DuoSet Ancillary Reagent Kit 2 (all from R&D Systems).
Fibroblast supernatants were assayed for Pro-collagen I using DuoSet ELISA Human Pro-Collagen I a1/COLIA1 kit, fibronectin using DuoSet Human Fibronectin and IL-6 using DuoSet Human IL-6 ELISA kit with the DuoSet Ancillary Reagent Kit 2 (all from R&D Systems).
3
Quantification of Plasma Biomarkers
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Secreted protein acidic rich in cysteine (also known as osteonectin) and FGF21 were quantified by single plex assays from MesoScale Discovery (R-plex; MSD, Maryland, United States), whereas IL-6, IL-8, IL-10, IL-13, IL-15, and IL-18 were quantified by multiplex assay (U-plex; MSD) in plasma obtained from venous blood samples taken from the arm of the participants. Intra-assay coefficients of variations for the standards for SPARC, FGF21, and the multiplex were 10.00, 8.02, and 8.28%, respectively. Samples were measured only once; therefore, their intra- and interassay coefficients of variations cannot be calculated. All antibodies, with the exception of SPARC, were validated for target specificity. Information can be found on the datasheet of the different U-plex antibody sets1 in the Supplementary Table S2 .
4
Cytokine release in whole blood
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The immediate differences in whole blood in-vitro evoked release levels of IL-1β and TNF-α after between the experimental and control group were the primary outcomes. Fasting heparinised samples of peripheral blood, taken between 08:00 and 09:00 A.M. and processed after 4 h, were used for whole blood culture. To induce the production of these cytokines, whole blood cultures were cultivated for 24 h at 37 °C in a humified 5% CO2 incubator with the presence of lipopolysacharide from Escherichia coli serotype 055:B5 (LPS; Sigma) at concentrations of 1 ng/ml (low dose stimulation) and 10 µg/ml (high dose stimulation). Following the incubation period, supernatants were centrifuged, aliquoted and frozen at − 80 °C until the analyses were performed. The levels of in-vitro IL-1β and TNF-α were determined using a custom-made U-plex (MSD, Maryland, United States) conforming to manufacturer recommendations. Supernatants were diluted 100-fold prior to testing.
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