Pe conjugated cd29

HUMSCs at passage three were analyzed using flow cytometry to examine the expression of pluripotent cell markers. Following trypsinization, ~1×10⁶ cells were pelleted, resuspended in phosphate-buffered saline (PBS) and fixed with 4% buffered paraformaldehyde (Gibco-BRL) for 20 min at room temperature. Cells were then incubated with monoclonal mouse anti-human antibodies against phycoerythrin (PE)-conjugated CD29 and CD59, or fluorescein isothiocyanate (FITC)-conjugated CD44 (BD Biosciences, Franklin Lakes, NJ, USA). Cells were incubated in the dark for 30 min at 4°C. In order to detect the presence of Oct-4, the cells were permeabilized in PBS with 1% Triton X-100 for 10 min at room temperature, fixed and incubated with a monoclonal mouse anti-human antibody against Oct-4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. Cells were then stained in the dark with a PE-conjugated secondary antibody for 30 min at 4°C. Control samples were incubated with FITC or PE-conjugated mouse IgG1 isotype antibodies (Santa Cruz Biotechnology, Inc.). Following incubation, the cells were washed with PBS, centrifuged at 200 × g for 10 min to remove any unbound antibodies, resuspended in 1 ml PBS and analyzed using EPICS XL flow cytometry (Beckman-Coulter Inc., Miami, FL, USA).