Quantification of Immune Mediators

Protein levels of galectin-1, galectin-3, IL-8, RANTES, and MIP-3α were simultaneously quantified using a custom-designed multiplex electrochemiluminescence (ECL) immunoassay, Sector Imager 2400 and Discovery Workbench Software (Meso Scale Discovery, Gaithersburg, MD), validated against traditional ELISA (9 (link), 37 (link), 38 (link)). Galectin-1 and -3 levels were also assessed by traditional commercially available ELISA (R&D Systems). The concentrations of soluble extracellular mediators released in the cell culture supernatants were measured in picograms/ml. The galectin levels in cell lysates (cell-bound galectins) were measured also in picograms/ml by ELISA and then normalized to the total protein of cell lysate, thus quantifying galectins in nanograms/mg total protein. Total protein levels were measured using the Pierce BCA protein assay. The ELISA and the BCA assays were read using a Victor2 reader (PerkinElmer Life Sciences).