Pe conjugated anti human cd73

Cells were washed with PBS twice then pre-blocked with 3% bovine serum albumin (BSA), or 2-5% serum from the same species, in PBS. The following direct antibodies were used: phycoerythrin (PE) mouse anti-human CD34 (1:20 from DakoCytomation, Barcelona, SP); FITC mouse anti-human CD45 (1:20 BD Pharmingen, Madrid, SP); FITC mouse anti-human CD105:
(1:100 from Serotec, Bavaria, Germany): PE-Cy5.5-conjugated mouse anti-human CD90 (1:20 from BD Pharmingen); PE-conjugated anti-human CD73 (1:20 from BD Pharmingen); and rabbit anti-human VEGF (1:20 from DakoCytomation). For detection of primary antibodies not directly labelled, the cells were washed with PBS, then incubated with polyclonal rabbit anti-mouse IgD/FITC Rabbit F(ab́) 2 (1:1,000 from DakoCytomation) for 30 min at room temperature. The stained cells were then washed twice with PBS and 1 × 10 5 cells were analyzed with a FACSAria flow cytometer (BD Bioscience, Madrid, SP). FACS data were generated by DIVA software (BD Bioscience). Negative control staining was performed using FITC-conjugated mouse IgG1k isotype, PE-conjugated mouse IgG1k isotype and PE-Cy5.5-conjugated mouse IgG1k isotype (all from BD Pharmingen).