Il 13

Buffy coats from healthy blood donors were obtained from the division of Clinical Immunology and Transfusion Medicine, Uppsala University Hospital (Uppsala, Sweden), and monocytes were isolated by gradient centrifugation and allowed to differentiate into macrophages with macrophage colony-stimulating factor (M-CSF) treatment for 6 days, as described previously (25 (link)). After macrophage differentiation, the macrophages were further differentiated into M1 macrophages through the addition of 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) plus 20 ng/ml IFN-γ for 48 h or M2 macrophages through the addition of 20 ng/ml IL-4 plus 20 ng/ml IL-13 (all from R&D Systems, Minneapolis, MN, USA) for 48 h. The differentiated M1 and M2 macrophages [the phenotypes were characterized as described previously (25 (link))] were washed twice with PBS and were cultured for another 48 h in RPMI 5% FCS (without either IFN-γ/LPS or IL-4/IL-13) to generate M1 and M2 CM. The collected CM was centrifuged to remove cell debris and stored in aliquots at −20°C.

HT-29 cells were transfected with 100 nM Pre-miR™ miR precursors (Negative control#1, miR-31, miR-155 or a combination of 50 nM miR-31 + 50 nM miR-155, Thermo Fisher Scientific) using Interferin (Polyplus, New York, NY, USA) following manufacturer’s instructions. For IL-13 stimulation experiments, cells were stimulated 24 h post-transfection with 100 ng/mL IL-13 (R&D Systems, Minneapolis, MN, USA) and harvested 24 h later.