Multiplex Cytokine Quantification

Fresh plasma was used for cytokine quantification using a customized 14-plex bead based flow cytometric assay (IncellKINE, IncellDx, Inc) on a CytoFlex flow cytometer as previously described using the following analytes: TNF-α, IL-4, IL-13, IL-2, GM-CSF, sCD40L, CCL5 (RANTES), CCL3 (MIP-1α), IL-6, IL-10, IFN-γ, VEGF, IL-8, and CCL4 (MIP-1β) (4). For each patient sample, 25 µL of plasma was used in each well of a 96-well plate. Samples were analyzed on a Beckman Coulter CytoFlex LX 3-laser flow cytometer using Kaluza Analysis Software (Beckman-Coulter, Miami, FL). All statistical analysis was performed using the Mann-Whitney test and a P value ≤0.05 was considered statistically significant.