8. CFSE staining-based lymphocyte proliferation assay

For testing lymphocyte proliferation, the procedures reported in a previous study were followed [26]. Briefly, 1×10⁶ PBMCs isolated freshly using Ficoll solution and suspended in 1 ml PBS containing 5% FBS were incubated with 5 μM CFSE at room temperature for 5 min. After thorough washing with PBS containing 5% FBS, the CFSE-labeled PBMCs were then co-cultured with BEAS-2B cells or hMSC1 in 5:1 ratio for 7 days at 37°C and 5% CO₂ in each well of a 12-well cell culture plate in RPMI-1640 complete medium containing 10 μg/ml PHA. After incubation, all lymphocytes were collected and the inhibition of lymphocyte proliferation was determined by gradual reduction of CFSE signal over the incubation period as detected by BD-Calibur flow cytometer. The data was further analyzed by FCS Express V3 software.