2 3 bis 2 methoxy 4 nitro 5 sulfophenyl 2h tetrazolium 5 carboxanilide xtt assay kit

The immunomodulation activity of the HUCMSCs was demonstrated with MLR. Human PBMCs from two different donors (male, 25 and 40 years old) who provided informed consent were isolated from heparinized blood by gradient centrifugation with Ficoll solution (Sigma-Aldrich) at 400 × g for 20 min at room temperature. Stimulator PBMCs were prepared by treatment with mitomycin (Sigma-Aldrich) at 25 μg/ml for 30 min at 37°C. Cell count and viability were assessed by trypan blue (Biowest) dye exclusion and then used directly in the MLR. The HUCMSCs were plated in triplicate at passage 2 into U-bottomed 96-well plates (Corning) at 10 5 cells/ ml in 100 μl of FCS-DMEM and allowed to adhere to the plate for 1 to 2 h. Human responder (10 5 PBMCs) and an equal number of stimulator PBMCs were added to the wells in 100 μl of RPMI-1640 (Invitrogen) in 10% inactivated FCS (Sigma-Aldrich). The cultures were incubated at 37°C in 5% CO 2 for 5 days, and the suspended responder PBMCs were then counted using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide (XTT) assay kit (Biological Industries). To test the role of HLA-G in MLR, 3 μg/well of HLA-Gneutralizing antibodies (87G Abs) or control antibody (20 μg/ml) (Exbio Antibodies, Praha, Czech Republic) was added on the first day of the MLR cultures.