PMC:7402624 / 65890-67795 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7402624","sourcedb":"PMC","sourceid":"7402624","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7402624","text":"Luminex\nPBMCs from patients were thawed and rested overnight at 37°C in complete RPMI (cRPMI, table S7). 96-well flat bottom plates were coated with 1 μg/mL of anti-CD3 (UCHT1, #BE0231, BioXell) in PBS at 4°C overnight. The next day, cells were collected and plated at 1 × 105/well in 100 μl in duplicate. 2 μg/mL of anti-human CD28/CD49d was added to the wells containing plate-bound anti-CD3 (Clone L293, 347690, BD). PBMCs were stimulated or left unstimulated for 16 hours, spun down (1200 rpm, 10 min) and 85 μL/well of supernatant was collected. Plasma from matched subjects was thawed on ice, spun (3000 rpm, 1 min) to remove debris, and 85 μl collected in duplicate. Luminex assay was run according to manufacturer’s instructions, using a custom human cytokine 31-plex panel (EMD Millipore Corporation, SPRCUS707). The panel included: EGF, FGF-2, Eotaxin, sIL-2Ra, G-CSF, GM-CSF, IFN-α2, IFN-γ, IL-10, IL-12P40, IL-12P70, IL-13, IL-15, IL-17A, IL-1Ra, HGF, IL-1β, CXCL9/MIG, IL-2, IL-4, IL-5, IL-6, IL-7, CXCL8/IL-8, CXCL10/IP-10, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, RANTES, TNF-α, and VEGF. Assay plates were measured using a Luminex FlexMAP 3D instrument (Thermofisher, Cat#APX1342).\nData acquisition and analysis were done using xPONENT software www.luminexcorp.com/xponent/). Data quality was examined based on the following criteria: The standard curve for each analyte has a 5P R2 value \u003e 0.95 with or without minor fitting using xPONENT software. To pass assay technical quality control, the results for two controls in the kit needed to be within the 95% of CI (confidence interval) provided by the vendor for \u003e25 of the tested analytes. No further tests were done on samples with results out of range low (\u003cOOR). Samples with results that were out of range high (\u003eOOR) or greater than the standard curve maximum value (SC max) were not tested at higher dilutions without further 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