Cd105 fluorescein isothiocyanate fitc

Immunophenotypic analysis of hASCs was performed at passages 4 and 10. The protocol was adapted from a previously published study [43 (link)]. Briefly, 5 × 10⁵ cells were incubated with 0.4 μg of each monoclonal primary antibody for 30 minutes at 4°C. After washing with PBS to remove unconjugated primary antibodies, the cells were incubated with Alexa Fluor 488 goat anti-mouse (Invitrogen) as a secondary antibody for 30 minutes at 4°C. Finally, the cells were washed with PBS and fixed with 200 μL 1% paraformaldehyde. As a control, hASCs were also incubated with only the secondary antibody to assess the background fluorescence. The following antibodies were used: CD90, CD166, CD45, CD19, CD105-fluorescein isothiocyanate (FITC), CD44 (HCAM), CD73-phycoerythrin (all from BD Biosciences, USA), CD54-FITC (ICAM; Caltag Medsystems, UK), CD34 (Santa Cruz Biotechnology, USA), HLA-ABC-FITC and HLA-DR-FITC (Abcam, USA). The hASCs were analyzed with a Guava® easyCyte™ 6-2 L Flow Cytometer (Millipore, USA) using Incyte acquisition software (Millipore). A minimum of 15,000 events were acquired for each experimental group. The data were analyzed with FlowJo 7.5.6 software(Treestar, Inc., USA ).