The human cytokines Interleukin 10 (IL-10), Transforming Growth Factor beta-1 (TGFβ1), Transforming Growth Factor beta-2 (TGFβ2), Transforming Growth Factor beta-3 (TGFβ3), and Tumor Necrosis Factor Alpha (TNFα) were purchased from Sigma-Aldrich, Interleukin 4 (IL-4) and Interferon Gamma (IFNy) from R&D Systems/Bio-Techne (R&D Systems/Bio-Techne, Wiesbaden-Nordenstadt, Germany), and Interleukin 6 (IL-6) and Vascular Endothelial Growth Factor165 (VEGF) from PeproTech (PeproTech, Winterhude, Germany). Porcine TGFβ3 was purchased from Biozol (Biozol, Eching, Germany), whereas porcine TGFβ1, TGFβ2, IL-4, IL-6, IL-10, IFNy and TNFα were from R&D System/Bio-Techne. Since there was no porcine VEGF available, the above mentioned human VEGF was also used for stimulation of pRMG.
To diminish the influence of cytokines present in FCS, both confluent pRMG and MIO-M1 cells were rinsed two times with prewarmed serum-free medium, followed by starvation for 1 h at 37°C and 5% CO2 with serum-deprived medium. Afterwards, cells were treated over night with IFNy, IL-4, IL-6, IL-10, TGFβ1, TGFβ2, TGFβ3, TNFα or VEGF165, respectively, in a randomized plate design at a concentration of 5 ng/ml in 2 ml medium without FCS. Untreated cells cultured in serum-free medium served as a control. For this study, cells were treated with each cytokine separately, but not with multiple cytokines in combination.
To diminish the influence of cytokines present in FCS, both confluent pRMG and MIO-M1 cells were rinsed two times with prewarmed serum-free medium, followed by starvation for 1 h at 37°C and 5% CO2 with serum-deprived medium. Afterwards, cells were treated over night with IFNy, IL-4, IL-6, IL-10, TGFβ1, TGFβ2, TGFβ3, TNFα or VEGF165, respectively, in a randomized plate design at a concentration of 5 ng/ml in 2 ml medium without FCS. Untreated cells cultured in serum-free medium served as a control. For this study, cells were treated with each cytokine separately, but not with multiple cytokines in combination.