The soluble plasma cytokines were quantified using the Luminex multiplex platform (Luminex, Austin TX) as described previously.10 (link) Briefly, the analytes were quantified using the Luminex multiplex platform with custom-developed reagents (R&D Systems, Minneapolis, MN), as described in detail43 (link) or single-plex ELISA (R&D Systems, Minneapolis, MN). The quantified analytes were read on MAGPIX® instrument and the raw data was analyzed using the xPONENT® software. Analytes quantified using single-plex ELISA were read using optical density. Values outside the lower limit of detection were imputed using 1/3 of the lower limit of the standard curve for analytes quantified by Luminex and 1/2 of the lower limit of the standard curve for analytes quantified by ELISA.
Single plex elisa
Manufactured by R&D Systems
Sourced in United States
The Single-plex ELISA is a laboratory equipment used for the detection and quantification of specific proteins or analytes in a sample. It is a plate-based assay technique that utilizes antibodies to capture and detect the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Luminex multiplex platform, by R&D Systems (1 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Multiplex elisa, by Mesoscale (1 mentions)
Custom developed reagents, by R&D Systems (1 mentions)
4 protocols using single plex elisa
1
Quantifying Soluble Plasma Cytokines
2
Proteomic Profiling of Tumor Cytokines
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Tumors (n = 6 mice/tumor type/time point) were flash-frozen in liquid N2 and stored at −80 °C until use. Tumors were next homogenized in lysis buffer containing 0.05% Tween-20 and protease inhibitors (Millipore-Sigma, Burlington, MA, USA). Protein concentrations were determined by bicinchoninic acid assay (ThermoFisher Scientific, Waltham, MA, USA). Proteomic analysis was performed using a Mouse Cytokine Chemokine 32-plex Magnetic Bead Panel (Millipore-Sigma, MCYTMAG-70-PX32) on a Bio-Plex 200 System (Bio-Rad, Hercules, CA, USA). Additional single-plex ELISA (R&D Systems) for intracellular adhesion molecule-1 (ICAM-1/CD54; DY796), IFNγ (DuoSet; DY485), vascular cell adhesion molecule-1 (VCAM-1/CD106; DY643), and TGF-β1 (MB100B/SMB100B) were performed according to manufacturer’s protocols.
3
Quantifying Inflammatory Cytokine Secretion
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Cells (2 × 104 per well) were cultured for 48 h and the supernatants harvested. Matrix Metalloproteinase 3 (MMP3) and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) were measured by single-plex ELISA (R&D Systems, Minneapolis, MN, USA), IP10, monocyte chemoattractant protein 1 (MCP1) and macrophage inflammatory protein 1 alpha (MIP1α) were measured via multiplex ELISA (Meso-scale discovery, Rockville, MD, USA).
4
Multiplex Quantification of Plasma Proteins
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Soluble proteins were quantified in EDTA-anticoagulated plasma using the Luminex multiplex platform (Luminex) with custom-developed reagents (R&D Systems), as described in detail (Guiducci et al., 2006 (link)) or single-plex ELISA (R&D Systems). Analytes quantified using the Luminex multiplex platform were read on the MAGPIX instrument and raw data were analyzed using the xPONENT software. Analytes quantified using single-plex ELISA were read using optical density. Values outside the lower limit of quantification were assigned a value of one-third of the lower limit of the standard curve for analytes quantified by Luminex and half of the lower limit of the standard curve for analytes quantified by ELISA. CXCL4 concentrations were determined using CXCL4/PF4 Quantikine (from R&D Systems) ELISA kit.
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