In Vitro Cytokine Stimulation

BMSCs (passage 4) were stimulated with 6.5 or 65 ng/ml of IFN-γ (R&D System Minneapolis, MN, USA), and with 1.5 or 15 ng/ml of TNF-α (R&D System Minneapolis, MN, USA), or the combination of IFN-γ and TNF-α (1.5 ng/ml of TNF-α + 6.5 ng/ml of IFN-γ or 15 ng/ml of TNF-α + 65 ng/ml of IFN-γ). After the adherent BMSCs were incubated with the cytokines for 48 hours, they were harvested using trypsin and were then evaluated by gene expression profiling. Prior to harvesting the BMSCs, the supernatant was collected, frozen and stored at −80 °C for protein analysis.