4.9. Assay for Quantification of Cytokine mRNA Expression

To quantify mRNA cytokine expression, THP-1 macrophages (M0) were obtained as described in Section 4.6. The interference of compounds with cytokine mRNA expression was obtained by treating the macrophages with the desired concentration of the compound for 6 h [30] in three different conditions: unstimulated, LPS-stimulated, and IL-4-stimulated. Untreated macrophages, stimulated or not, were also used. After incubation, supernatants were aspirated and cells were washed. The mRNA was isolated using TripleXtractor reagent, and samples were stored at −70 °C until use. After separation of the RNA fraction, the samples were purified using the GRS Total RNA Kit (Blood and Cultured Cells). RNA concentration and purity were assessed by the NanoDrop Lite spectrophotometer (Thermo Scientific^®, Waltham, MA, USA). mRNA samples were the template for cDNA synthesis, using the High Capacity RNA-to-cDNA Kit. The reactions were performed in the StepOneTM qPCR Real-Time PCR instrument containing 1 × Master Mix, 1 × probes (TaqMan^® Gene Expression assays IL-1β, Hs01555410_m1; TNF-α, Hs02621508_s1; IL-10, Hs00961622_m1; and TGF-β1, Hs00998133_m1—all from Applied Biosystems, Foster City, CA, USA). The housekeeping gene B2M (TaqMan^® Hs99999907_m1; Applied Biosystems, Foster City, CA, USA) served as endogenous reference, expressed at a consistent level to normalize the results. The data analysis was carried out using the StepOneTM Software v2.2 (Applied Biosystems, Foster City, CA, USA) with the same baseline and threshold set for each plate to generate quantification cycle values (Cqs) for all the mRNAs in each sample.