Luminex magpix system

HCT116-GFP (5000 cells/100μL/well) were seeded in 96-well Nunc plates and co-cultured with unstimulated PBMC (i.e., no addition of anti-CD3 or IL-2). Co-cultures were treated with DMSO vehicle (0.1%), OXP (10 μM), or FTD (3 μM, 10 μM) for 48 h before supernatants were collected. Cytokine levels in the supernatants were measured using a Bio-Plex Pro Human Cytokine 8-plex assay (Bio-Rad, USA) according to the manufacturer’s instructions. Briefly, the cytokines of interest are bound to magnetic beads via antibodies and subsequently detected using biotinylated antibodies with a fluorescent reporter. Fluorescence was measured using a Luminex MAGPIX system (Bio-Rad, USA) and concentrations were calculated using standard curves.

The supernatant content of cytokines were measured using the Luminex/MagPix system and commercially available kits for various analytes (Biorad, Hercules, CA, USA; R&D, Minneapolis, MN, USA; Millipore, Burlington, MA, USA). The assay is based on binding of the target of interest via antibodies to magnetic beads. The target is detected using biotinylated antibodies with a fluorescent reporter. The assays were performed according to the manufacturer instructions. Briefly, for the cytokine assay, the supernatant samples were incubated first with beads, then with detection antibody and finally with streptavidin-PE. The fluorescence was measured using the MagPix instrument (BioRad) and the concentration levels were calculated by fitting a standard curve.

A panel of biochemical markers associated with either bone or energy metabolism was measured in serum to test for possible metabolic established effects of the training protocol. The biomarkers were tested through a Milliplex^® multiplex immuno-based fluorescent Luminex ^®assay (EMD Millipore, Billerica, MA, USA) on a Luminex MagPix^® system (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, the assay allows the measurement of multiple analytes within the same well, containing the same sample aliquot through the addition of magnetic fluorescent beads coated with antibodies against the specific analyte of interest, thus completely avoiding inter-assay variability. The analytes, and the relative intra- (CV_w) and inter-assay (CV_b) variability, were: interleukin (IL)-1β (7%, 9%), tumor necrosis factor (TNF)-α (8%, 7%), DKK-1, sclerostin (SOST; 6%, 13%), osteocalcin (OCN; 5%, 12%), osteopontin (OPN; 2%, 12%), osteoprotegerin (OPG; 5%, 11%), parathyroid hormone (PTH; 4%, 9%), insulin (6%, 8%), and leptin (5%, 11%).

A panel-relevant proteins identified by gene and protein arrays of pooled samples has been assayed and validated in each single population of NP, AF, and EP cells of seven donors bearing the most responsive Ff genotype.
A custom panel (Human Magnetic Luminex Screening Assay, Bio-Techne, Minneapolis, MN, USA) for the multiple detection of BMP-2, BMP-7, IL-1Ra, IL-2, IL-3, IL-6, IL-17A, MMP-1, MMP-3, and MMP-13 was created. Detection range and sensitivity for these analytes are reported in Supplementary Table S2. A further fixed panel of 17 analytes (Human Discovery Th9/Th17/Th22 Fixed Panel, Bio-Techne) comprising IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p70, IL-13, IL-15, IL-17A, IL-17E, IL-33, MIP-3α (CCL20), CD40 Ligand, GM-CSF, IFNγ, TNFα was performed to validate some previously analyzed parameters and to add other 14 proteins to the validation, using a more sensitive methods in comparison with a protein array. The range of detection of each protein in the fixed panel is in the order of pg/mL and is reported on the product datasheet.
The assay was performed on a MagPix™ Luminex System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the manufacturer instructions.

A panel of 14 myokines known to be released by skeletal muscle during physical activity were assayed through a high sensitivity multiplex bead-based immunofluorescent assay (Myokine Magnetic Bead Panel, Millipore, Burlington, MA, United States) on a MagPix™ Luminex System (Bio-Rad Laboratories, Inc., Hercules, CA, United States) following manufacturer instructions. The following specific myokines were tested in plasma samples: apelin, fractalkine, brain-derived neurotrophic factor (BDNF), osteonectin (SPARC), leukemia-inhibitory factor (LIF), interleukin (IL)-15, myostatin/growth differentiation factor (GDF)8, fatty acids binding protein (FABP)3, follistatin-like protein (FSTL)-1, oncostatin M (OSM), IL-6, fibroblast growth factor (FGF)21, and osteocrin/musclin. Furthermore, plasma irisin concentrations were measured by a competitive enzyme immunoassay (Phoenix Pharmaceuticals, Inc., Burlingame, CA United States), having a sensitivity of 1.29 ng/ml. All samples were tested in duplicate.