Luminex assay human premixed multi analyte kit

Serum cytokines and chemokines were assessed according to the instruction of Luminex^® Assay Human Premixed Multi-Analyte Kit (R&D Systems, Inc. Minneapolis, MN, USA). The kit detects BAFF, CCL3, CCL11, CXCL10, IFN-γ, IL-2, IL-5, IL-8, IL-12 p70, CCL2, CCL4, CXCL1, GM-CSF, IL-1β, IL-4, IL-6, IL-10, IL-13, TNF-α, CXCL12 and RANTES. Briefly, 50 µl of standard or sample was added to each well of a microplate followed by the addition of 50 µl of diluted Microparticle Cocktail. The microplate was then covered with a foil plate sealer and incubated on a horizontal orbital microplate shaker at 800 rpm at room temperature for two hours. The plate was washed three times with Wash Buffer. Then, 50 µl of diluted Biotin-Antibody Cocktail was added to each well and incubated again on a horizontal orbital microplate shaker at 800 rpm at room temperature for one hour with a foil plate sealer. After that, the plate was washed 3 times, 50 µl of diluted Streptavidin-PE were added to each well, and the plate was incubated on a horizontal orbital microplate shaker at 800 rpm at room temperature for 30 minutes with a foil plate sealer. Finally, the plate was washed three times, 100 µl of Wash Buffer were added to each well, and the plate was incubated for 2 minutes at room temperature. The samples were then read in a Luminex^® MAGPIX^® analyzer (R&D Systems, Inc. Minneapolis, MN, USA).

The dishes were incubated at 37 ℃, 5% carbon dioxide (CO2) [21 (link)]. The amount of growth factors released in the culture media were sampled at 15 min, 1 day, 3 days, 7 days and 14 days [14 (link), 21 (link)]. In reference to a previous study, 5 ml of culture media was collected at each time point, frozen at − 80 ℃ and replaced with 5 ml of additional culture media [14 (link)]. Measurement of growth factors was performed by ELISA kits (Luminex® Assay Human Premixed Multi-Analyte Kit, R&D Systems, Inc., Minneapolis, USA and TGF-β1 Single Plex Magnetic Bead Kit, EMD Millipore, Darmstadt, Germany). ELISA was performed at Filgen, Inc. (Nagoya, Japan). PDGF-AB, VEGF, TGF-β1, bFGF, and SDF1 were chosen to be analyzed since the growth factors have been shown to be favorable in meniscal healing and have been detected to be released from platelet concentrates [12 (link), 13 (link)].

Luminex® Assay Human Premixed Multi-Analyte Kit (R&D systems) was used for simultaneous measurement of IL-1ß, IL-1RA, TNF-α, IL-8, MCP-1 and ICAM-1 (negative control). The plasma samples were diluted with PBS prior to analysis and analyzed in duplicates according to the manufactures instructions. The chemiluminescense was read on a VERSAmax Absorbance Microplate Reader (Molecular Devices). Final plasma concentrations were calculated using the Bioplex software supplied by the manufacturer. The standard curve was constructed using a 4-parameter logistic function.