Cd90 apc vio770

The antigenic phenotype of UCB cells was assessed before and during ex vivo expansion. Approximately 1 × 10⁵ cells per test were incubated with the required antibody panel as per manufacturer’s instructions for 30 min at room temperature. All antibodies were preconjugated and monoclonal. Progenitor panel: CD34-PerCP-Vio700, CD38-FITC, CD45RA-VioGreen, CD90-APC-Vio770, CD133-APC, CD135-PE-Vio770 (Miltenyi Biotech) and CD33-PE (BD Biosciences). Mature stage myeloid panel: CD13-APC, CD14-PE, CD15-VioGreen, CD34-PerCP-Vio700, CD33-APC-Vio770, CD38-FITC, CD123-VioBlue (Miltenyi Biotech). Analysis was conducted with CITRUS (cluster identification, characterisation and regression) software [31 (link)]. The software uses a regularized supervised learning algorithm to determine the populations and features of the samples being analysed in a correlative manner. The results of a CITRUS run are clusters (populations) that differentiate the observed endpoint of the samples, and the features (relative population abundance or median expression of a functional marker) of the clusters that are responsible. All CITRUS analyses were conducted using a Significance Analysis of Microarrays (SAM) association model and a minimum cluster size of 4%, at least 5000 events from each sample were included for the analysis.

Cell surface marker expression was evaluated using the following primary antibodies: the stage-specific embryonic antigen 4-PE (SSEA4-PE; BioLegend, Dedham, MA, USA), CD73-APC (Thermo Fisher Scientific), CD90-APC-Vio770 (Miltenyi Biotec, San Diego, CA, USA), CD105-PerCP-Vio700 (Miltenyi Biotec), and CD146-PE (Thermo Fisher Scientific). Expanded cells (2 × 10⁵ each group) were first blocked through a 30 min incubation in cold phosphate-buffered saline (PBS) containing 0.1% ChromPure Human IgG whole molecule (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) followed by incubation with primary antibodies at 4 °C for 30 min. Fluorescence was evaluated by a FACS Calibur (BD Biosciences) using the FCS Express software package (De Novo Software).