Procartatm immunoassay

Immune molecules in plasma were previously measured using a magnetic bead-based 61-plex immunoassay (customized ProcartaTM immunoassay, Affymetrix) [26 (link)]. The immune profiling of extracellular vesicles was performed at the Human Nutritional Chemistry Service Laboratory at Cornell University using a human 48-plex magnetic bead kit (Bio-Plex Pro Human Cytokine Screening Panel, 48-plex, Bio-Rad). Prior to analysis, EV samples were treated with Triton 1% to allow the release of encapsulated cytokines [32 (link)]. Each sample was measured in duplicate on a MAGPIX^® Multiplexing System (Luminex Corp.). For each well, we used the median fluorescence intensity of all beads measured for a given analyte and averaged the two replicates and results were accepted when the coefficient of variation (CV) was below 15%.

A magnetic bead-based 61-plex immunoassay (customized Procarta^TM immunoassay, Affymetrix) was used to measure plasma concentrations of immune molecules (Additional file 1: Table S3A). Case and control plasma samples were coded, randomized, and run in duplicate along with serial standards, buffer controls, and in-house human control plasma samples [53 (link)]. Mean fluorescence intensities of analyte-specific immunoassay bead sets were detected by flow-based Luminex™ 200 suspension array system (Luminex Corporation, Austin, TX) [54 (link)]. Cytokine concentrations were calculated by xPONENT (build 3.1.971.0) and Milliplex Analyst^TM software (v.5.1.0.0) using a standard curve derived from known reference concentrations supplied by the manufacturer. A five-parameter model was used to calculate final concentrations by interpolation. Values are expressed in pg/ml. Concentrations obtained below the sensitivity limit of detection (LOD) of the method were recoded to the mid-point between zero and the LOD for that analyte for statistical comparisons. Values obtained from reading of samples that exceeded the upper limit of the sensitivity method were further diluted and cytokine concentrations calculated accordingly. Feature scaling (data normalization) was used to standardize the range of cytokine values for the MeV heatmap [55 ], and log-transformation was used for network analysis.