Cd19 ecd

The ability of MSCs to differentiate into osteoblasts and adipocytes was confirmed prior to use. Osteogenic differentiation was evaluated by calcium deposition staining using the Alizarin Red staining. The induction of adipogenic differentiation was apparent by intracellular accumulation of lipid-rich vacuoles that stained with Oil Red O. The specific surface molecules of HUCMSCs were characterized by flow cytometric analysis. The cells were stained with the following antibodies: CD14-FITC, CD19-ECD, CD29-FITC, CD34-PE, CD44-FITC, CD45-FITC, CD73-PE, CD90-FITC, CD105-PE, HLA-DR-FITC (BD Pharmingen, USA). Thereafter, the cells were analyzed using a Becton Dickinson flow cytometer (Becton Dickinson, San Jose, CA).

After PBMC purification and NK cell quantification, 3 million cells were incubated at 37°C for 4h or overnight with K562 target cells at an Effector (NK cell): Target ratio of 1:10 in a final volume of 500μl (RPMI Glutamax with 10% FBS and 10u/ml IL2). The medium also contained 1.5μl anti-CD107a antibody (BD Biosciences, Franklin Lakes, NJ) and 1μl monensin to prevent CD107a degradation (BD Golgi-Stop BD Biosciences). Then, cells were resuspended in 50μl of an antibody cocktail containing 7AAD, the anti-CD45RO-FITC, -CD69-PE, -CD19-ECD, -CD56-PECy7, -CD3-APC, -CD45RA-APCAlexaFluor750, -CD107a-HV500 and -CD16-KO antibodies (BD Biosciences, Beckman). Samples were analyzed on a Beckman Coulter FACS Gallios flow cytometer using the Kaluza software. Events were initially gated on forward and side scatter (SSC) to identify lymphocytes. A bivariate plot of CD56 versus CD3 was used to acquire at least 10,000 NK cells.