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. 2013 Dec 1;2(6):431–439. doi: 10.1089/biores.2013.0040

FIG. 3.

FIG. 3.

Effect of MTX and AZA on ASC-mediated inhibition of T-lymphocyte proliferation. (A) CFSE-labeled PBMCs were stimulated with anti-CD3/CD2/CD28-coated beads in the absence or presence of ASCs (ratio 1:25 ASC:PBMC), MTX (100 μg/mL), AZA (10μM), or a combination of ASCs and drugs. Proliferation of the viable population of CD3+ cells (CD3+/7AAD− cells) was monitored after 120 h by flow cytometry, and the percentage of cells per generation was determined using FCSExpress software. Mean and standard deviation of the percentage of cells accumulated on each generation is shown. N=8. Similar results were obtained when combination of ASCs with MTX (0.01 μg/mL) and AZA (1 μM) were used. (B) The immunosuppressive capacity of ASCs pretreated with the drugs was studied. ASCs were cultured for 7 days in medium alone or with MTX (100 μg/mL) or AZA (10 μM). Then, PBMCs were stimulated in the presence of control or MTX- or AZA-pretreated ASCs (ratio 1:25 ASCs:PBMCs) and proliferation of the viable population of CD3+ cells was monitored after 120 h by flow cytometry. Mean and standard deviation of the percentage of cells accumulated in each generation is shown. N=8. Similar results were obtained when ASCs were pretreated with MTX (0.01 μg/mL) and AZA (1 μM). *p=0.043. CFSE, carboxyfluorescein diacetate N-succinimidyl ester; PBMCs, peripheral blood mononuclear cells.