Determination of Cytokine Levels

The cytokines IL-4, IL-6, IL-8, IL-10, MIF, and TNF-α were assessed in BeWo or HTR-8/SVneo cells supernatants. The molecules were measured by sandwich enzyme-linked immunosorbent assay (ELISA: BD Biosciences, San Diego, CA, United States; or R&D Systems, Minneapolis, MN, United States) and data were expressed in pg/ml. Besides, MIF, IL-4, IL-8 e IL-6 cytokines were measured in supernatants of villous explants, according with manufacturer’s instructions. For villous explants, the data were normalized according to the protein concentration of each villous as described above and obtained by the ratio between concentration of cytokines in pg/ml and concentration of total protein from Bradford assay in μg/ml, resulting in pg/μg of tissue. The limits of detection of each cytokine were determined from standard curves as follow: IL-6 = 4.7, IL-8 = 0.8, TNF-α = 7.8, MIF = 7.8, and IL-10 = 7.8 (all in pg/ml).