Pe conjugated cd73

Cells harvested from 3rd passage (P3) were used for immunophenotyping analysis. Mouse anti-human monoclonal antibodies: phycoerythrin (PE)-conjugated CD73, CD90, CD105, CD45, CD11b, CD34, CD79a, and FITC-conjugated HLA-DR (BD Biosciences, San Jose, CA) were used. As a control, isotype PE-conjugated IgG1 and FITC-conjugated IgG2a (BD Biosciences) were used. The cell suspension, containing 1 × 10⁶ cells, were incubated with monoclonal antibodies at room temperature in the dark and fixed with BD Cytofix^TM (BD Biosciences). The samples were analyzed on FACSCalibur cytometer (BD Biosciences) and the resulting data were processed using CellQuest^TM Proversion 6.0 software (BD Biosciences).

Cultured cells (passage 1) were trypsinized and stained with a panel of antibodies for fluorescence-activated cell sorting (FACS) analysis. Approximately, 1 × 10⁵ cells were re-suspended in phosphate buffered saline (PBS) and incubated with IgG block for 5 minutes to block non-specific binding. The following antibodies were used: AF-700 conjugated CD3 (BD BioSciences, USA), PE conjugated CD14 (BD, Immunocytometry, USA), APC conjugated CD19 (BD BioSciences, USA), PE conjugated CD34 (BD, BioSciences, USA), APC conjugated CD44 (BD, Pharmingen, USA), FITC conjugated CD45 (BD Pharmingen, USA), PE conjugated CD73 (BD Pharmingen, USA), AF-700 conjugated CD90 (Biolegend, USA) and APC conjugated CD105 (Biolegend, USA). Cells were stained for 30 minutes at 4°C with the antibodies. After washing, samples were analyzed on a LSR II flow cytometer (BD, USA) and at least 10,000 events were acquired for each population. Data acquisition and analysis were performed using FACS DIVA software (BD Biosciences, USA). Unstained cells were used to establish flow cytometer settings. Debris and cells/particles with auto-fluorescence were removed by using a threshold on the forward scatter.