To assess the effects of MenSCs on CD4 + T cells proliferation, CD4 + T cells were co-cultured in different settings as mentioned above. Before co-culturing, CD4 + T cells were washed and labeled with 5 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, USA).
To investigate the effects of MenSCs secretome on CD4 + T cell proliferation, a transwell system (Corning, USA) was utilized. Briefly, IFN-γ/IL-1β-treated MenSCs were seeded in the lower chamber of a 24-well transwell plate. Later, CD4 + T cells were added into the upper chamber and plates were incubated for five days in incubator. In some settings, 1 mM 1-Methyl-DL-tryptophan (Sigma, USA) as IDO blocker, 20 mM Indomethacin (Sigma, USA) as PGE2 inhibitor, 10 µg/mL anti-IL-6 antibody (R&D systems, USA), 10 µg/mL anti-IL10 antibody (R&D systems, USA) or 10 µg/mL anti-TGF-β (R&D systems, USA) were added to the co-cultures in order to investigate the involvement of aforesaid molecules on modulatory effects of MenSCs on T cell proliferation or Treg generation. After 5 days, proliferation of CD4 + T cells and frequency of Tregs were assessed by flow cytometry.
To investigate the effects of MenSCs secretome on CD4 + T cell proliferation, a transwell system (Corning, USA) was utilized. Briefly, IFN-γ/IL-1β-treated MenSCs were seeded in the lower chamber of a 24-well transwell plate. Later, CD4 + T cells were added into the upper chamber and plates were incubated for five days in incubator. In some settings, 1 mM 1-Methyl-DL-tryptophan (Sigma, USA) as IDO blocker, 20 mM Indomethacin (Sigma, USA) as PGE2 inhibitor, 10 µg/mL anti-IL-6 antibody (R&D systems, USA), 10 µg/mL anti-IL10 antibody (R&D systems, USA) or 10 µg/mL anti-TGF-β (R&D systems, USA) were added to the co-cultures in order to investigate the involvement of aforesaid molecules on modulatory effects of MenSCs on T cell proliferation or Treg generation. After 5 days, proliferation of CD4 + T cells and frequency of Tregs were assessed by flow cytometry.