Optmizer media

CD3⁺ T lymphocytes were isolated from umbilical cord blood samples (Canadian Blood Services, Montreal, QC, Canada) using a positive selection kit (StemCell Technologies, Vancouver, BC, Canada) and stained with 1 µM CFSE (Molecular Probes, Thermo Fisher, Burlington, ON, Canada) for 5 min. Cells were washed twice with PBS (Invitrogen, Thermo Fisher, Burlington, ON, Canada), resuspended in OpTmizer media (Invitrogen), and 4 × 10⁴ cells per well were seeded in triplicate in a 96 wells plate (Life Science, Corning, Tewksbury, MA, USA) and stimulated with CD3/28 Dynabeads (Invitrogen). MSCs were grown as described above in proinflammatory conditions induced with TNFα and IFNγ, 48 h prior to harvesting. MSCs were resuspended in OpTimizer media and 4 × 10³ cells were added to the stimulated T cells. Co-cultures were placed at 37 °C in a humidified incubator, at 5% CO₂ and 5% O₂, for five days. To analyze T lymphocyte proliferation, cells were stained with anti-CD45 antibodies (clone 2D1) conjugated to AlexaFluor700 (eBioscience, Thermo Fisher, Burlington, ON, Canada) and 10 ng µL⁻¹ propidium iodide (Sigma) and analyzed using a BDFortessa flow cytometer. Stimulated CFSE fluorescence of CD3⁺CD45⁺ cells, in the absence or presence of MSCs, were compared to determine T cell proliferation.