Multiplex Cytokine Profiling Across Samples

Measurement of cytokines and soluble factors is considered reflective of the overall basal immune microenvironment. Multiplexing was done using the Meso Scale Discovery (MSD) human V-Plex multi-spot assay kits, which is designed to reduce sample handling and allow for simultaneous processing of multiple samples and analytes, hence reduce variability (28 ). In brief, 25 µl of prepared calibrators and samples were added to the wells in duplicates and the plates were processed according to manufacturer’s instructions (28 ). After processing, the plates were read on an MSD Quickplex SQ120 imager, and concentrations interpolated from a standard curve. The respective upper (ULOQ) and lower (LLOQ) limits of quantification for each analyte were also inferred. Analyte concentrations below the LLOQ were assigned half the LLOQ concentration, whilst those above the ULOQ were assigned the ULOQ value, as previously reported (29 (link)). Duplicate observations were aggregated into a mean value and used in downstream analysis.