Flow cytometric and confocal microscopy analysis of MSC surface adhesion molecules

After 2 days culture, dissociated 2D and 3D cultured MSCs were incubated with mouse anti-human fluorescein isothiocyanate (FITC) conjugated anti-CXCR4, rat anti-human FITC conjugated anti-CD44, mouse anti-human FITC conjugated anti-ICAM-1 and mouse anti-human FITC conjugated anti-VCAM-1 (all at 1:100; all from eBiosciences). After washing and resuspending in FACS buffer, 10,000 events were analysed flow cytometrically (FACSCalibur; Beckman Coulter) with data visualised using Flowing software 2.5.1 (University of Turku, Finland). Adhesion molecule expression was presented as a percentage of the total cell count. Confocal analysis of the CD44 and ICAM-1 was also performed on formalin fixed MSCs as previously described using rat anti-human FITC conjugated anti-CD44 and mouse anti-human FITC conjugated anti-ICAM-1 (all at 2:25 dilution; eBiosciences) (27). A laser scanning confocal microscope (Zeiss LSM Zen 780, USA) with a 40x magnification water immersion lens was used to scan slides and obtain images for qualitative analysis.