To characterize hAD-MSCs, they were cultured to passage 3 and then resuspended in PBS and incubated for 30′ at 4°C with the following antibodies: phycoerythrin-conjugated anti-CD34, phycoerythrin-conjugated anti-CD90 (Abcam), fluorescein-conjugated anti-CD45, fluorescein-conjugated anti-CD105 (Chemicon), fluorescein-conjugated anti-IDO (R&D Systems), fluorescein-conjugated anti CD80 (Biolegend), phycoeritrin-conjugated anti CD40 (Abcam), Peridinin Chlorophyll Protein Complex (PerCP)-conjugated anti HLA-DR (Miltenyi) and Allophycocyanin-conjugated CD86 (Miltenyi). For intracellular markers (IDO) a previous permeabilization step was performed. For nitric oxide (NO) detection, a DAF-FM diacetate kit (Molecular Probes) was used. Briefly, detached cells were incubated with DAF-FM diacetate for 30′ (hMSCs) or 60′ (rbMSCs) at 37°C, followed by 30′ of PBS. Cells were then resuspended in PBS and analyzed in a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using the Cell Quest Pro program.
For AD-MSCs stimulation, they were cultured with 20 ng/ml IFN-γ and TNF-α for 4 h. Then the medium was changed and cells were cultured without cytokines for another 16 h prior to FACS analysis.
For AD-MSCs stimulation, they were cultured with 20 ng/ml IFN-γ and TNF-α for 4 h. Then the medium was changed and cells were cultured without cytokines for another 16 h prior to FACS analysis.