*3.7. Cytotoxicity Assessment*

DCs were treated with genistein (2.5, 5, 10, 20, and 40 μM) for 24 h. The cells were then measured in terms of their cell viability by the CCK-8 assay (Sigma), according to standard protocols, as described previously [32]. Triplicate treatments were performed for each sample in all experiments.

#### *3.8. Analysis of DC Maturation*

Maturation was determined by measuring the upregulation of MHC class II and three co-stimulatory molecules (CD40, CD80, and CD86), as described previously [31,32]. DCs were untreated or treated with LPS (100 ng/mL) or LPS + genistein (5, 10, and 20 μM) for 24 h. Cell aggregation was examined by microscopy (40×). Then, the cells were stained with monoclonal antibodies (mAbs), specific to mouse CD11c, MHC class II, CD40, CD80, and CD86 (Biolegend), and analyzed by flow cytometry. The fluorescence intensity of MHC class II, CD40, CD80, and CD86 was determined, following gating with a forward side scatter (FSC) and CD11c+ expression. The change in the mean fluorescence intensity (MFI) from LPS alone to LPS + genistein was indicated.
