Briefly, 100 μl of detection antibody was added to all wells, except blank, mixed gently and incubated overnight (16–24 h) at 4 °C. Plates were washed 3 times and standards and supernatant were added in the respective wells in duplicate. After the incubation time, the plates were washed again and incubated with 200 μl of conjugate for 60 min at room temperature. Plates were washed 3 times again and 200 μl of substrate was added and incubated for 15 min at room temperature in the dark. Belnacasan The reaction was stopped by the addition of 50 μl stop solution, and colour was measured in an automated microplate spectrophotometer (Epoch, Biotek,
Winooski, VT, USA). The total amounts of cytokines were determined as picograms (pg/ml). Results were calculated using the standard curves created in each assay. The ELISA assays were carried out
in a blind fashion in triplicate. The morphological findings of the in situ-like neoplasic areas, in each period, are depicted in Fig. 1. At 3 days, few small colonies of carcinoma epithelial cells were observed surrounded by numbered myoepithelial cells that assumed polyhedral p53 inhibitor and stellate morphology. At 5 and 7 days of culture, the number of carcinoma epithelial cells was more abundant assuming a cluster forming. Moreover, after 9 days of cell culture, the malignant epithelial cells were predominant in the plate, and few myoepithelial cells were visualized in the cell culture. Under the malignant cell carcinoma stimulation, the myoepithelial cell assumed a spindle-shaped morphology. Almost no myoepithelial cells were observed after the 13th day of cell culture and after 16 days, no in situ-like area was observed and there was a predominance of malignant cell demonstrating that the myoepithelial cells were not able to suppress the tumour cells blocking the malignant
cell proliferation. The IL-6 levels were higher when compared with IL-4 and IL-10 cytokines, in all studied periods (Fig. 2). IL-6 was over Selleck C59 expressed since the beginning of the cell culture and reached the peak after 9 days of cell culture (Fig. 2B). Interesting, the peak of IL-6 release fitted with the predominance of malignant cells in the culture when co-cultured with myoepithelial cells. After that, the levels started to decrease mainly at 16 days. Isolated, the myoepithelial cells released higher levels of IL-6 than the malignant cells which just produced IL-6 at the beginning of the cell culture. On the other hand, IL-4 secretion was the lowest in comparison with the other cytokines (Fig. 2A). In the in situ like areas, the IL-4 secretion was elevated at the beginning of the cell culture where was predominant the benign myoepithelial cells.