All cell culture procedures were performed under sterile conditions in a Class II laminar biohazard safety cabinet (ESCO). Cell cultures were incubated at 37°C in humidified 5% CO2 incubators (RSBiotech). MSCs were cultured in complete MSC medium consisting of Dulbecco's modified Eagle's medium with F-12 (HAM) nutrient mixture [1:1] (DMEM/F12). with GLUTAMAX -I (Gibco, Invitrogen, USA), supplemented with 10% pre-selected fetal bovine serum (Stem Cell Technology Inc.), 1% penicillin/streptomycin (Gibco, Invitrogen), 0.5% Fungizone (Gibco, Invitrogen), 0.1% gentamicin (Gibco, Invitrogen), with or without 40 ng/ml basic fibroblast growth factor (bFGF) (Peprotech, USA). The serum used was pre-selected by the manufacturer to support optimal growth of MSCs in vitro while preserving the multi-potency of MSCs to differentiate into chondro-, adipo- and osteogenic pathways.3.3 T cell media PBMCs were cultured in a complete T cell medium composed of RPMI 1640 (Gibco BRL, Invitrogen) supplemented with 10% fetal bovine serum (Gibco BRL, Invitrogen) or human AB serum and 1% penicillin/streptomycin (Gibco BRL, Invitrogen), 0.5% Fungizone (Gibco BRL, Invitrogen), 0.1% Gentamicin (Gibco BRL, Invitrogen). 3.4 Flow cytometry analysis 3.4.1 Immunophenotyping Cell surface markers were determined by direct immunofluorescence staining and analyzed by flow cytometer. All antibodies were listed in Table 1. Cells were harvested and washed with 0.5% BSA in 1X PBS (phosphate-buffered saline). Aliquots of 105-106 cells were labeled with anti-human monoclonal antibodies for 20 minutes at 2-8°C and washed with 0.5% BSA in 1X PBS. All antibodies are produced in mice against human epitopes. Fluorochrome analysis was middle of paper......Tritium thymidine incorporation tests (3H-TdR) Proliferation tests were measured by tritium thymidine incorporation (3H-TdR) which reveals the proportion of cells in the S phase of the cell cycle. Briefly, after 18 hours of incubation, 3H-TdR (0.037 MBq/well [0.5 µCi/well]) (Pelkin Elmer) was added to pulse the cultures. At the end of incubation, cells were harvested on A-glass fiber filter mats (Perkin Elmer) using a 96-well plate manual cell harvester (MACH IIIM-FM, Tomtec, Inc. Hamden, CT USA). A scintillation cocktail was added to amplify the signal. Liquid scintillation spectroscopy was counted using the Microbeta Trilux beta counter (Pelkin Elmer). 3.6 Statistical analysis Values ​​of all measurements are presented as mean ± standard deviation or otherwise indicated. Comparisons for all pairs were performed by Student's t test. Significance levels were set at the p value of 0.05.