Beckman cytomics fc 500 flow cytometry analyzer

The 4th passage of BMSCs, PB-MSCs, and USCs was harvested by 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA). For each staining, about 1 × 10⁶ cells were incubated with fluorescein isothiocyanate (FITC) or phycoerythrin- (PE-) conjugated monoclonal antibodies: CD29, CD34, CD45, CD73, CD90, CD105, CD166, and HLA-DR (BD Pharmingen™) at 4°C for 30 min in the dark room and then washed and resuspended in 200 μl PBS (pH = 7.4). The phenotypic analysis was performed with a Beckman Cytomics FC 500 Flow Cytometry Analyzer (Beckman Coulter, USA). CD31 expression after endothelial differentiation was tested under the same procedure.

When at passage 4 (P4), hUSCs were harvested using trypsin-EDTA, and 1 × 10⁶ hUSCs were resuspended in Hank's Balance Salt Solution (HBSS) supplemented with 1% (v/v) bovine serum albumin (BSA). Cells were incubated for 30 min at 4°C in the dark and followed by the following monoclonal antibodies: CD34-APC (BD, USA), CD45-PE (BD, USA), HLA-DR-PE (BD, USA), CD29-PE (BD, USA), CD73-PE (BD, USA), CD90-FITC (BD, USA), CD105 (Abcam, UK), and CD166 (Abcam, UK). Next, cells were washed with PBS and incubated with the appropriate secondary antibodies. Cells were analyzed using the Beckman Cytomics FC500 Flow Cytometry Analyzer (Beckman Coulter, USA).