Cd146 pe vio770

The expressions of antigens associated with hematopoietic, cardiomyocyte, and endothelial lineages were evaluated. Human control and differentiated CD34⁺ cells were dissociated with 0.25% Trypsin-EDTA (Life Technologies, Courtaboeuf, France) for 4 min at 37 °C, washed, counted, suspended in PBS, and stained for 15 min at room temperature with different combinations of the following mouse anti-human monoclonal antibody surface markers: FITC-CD34 (130-113-178), APC-CD309 (KDR) (130-093-601), CD73-PE (130-112-060), CD133-PE (130-098-826), CD90-PE (130-117-537) (Miltenyi Biotec, Bergisch Gladbach, Germany), CD106-APC (305809) (Biolegend, Amsterdam, The Netherlands), CD105-VioBlue (130-112-320), CD117-PE-Vio770 (130-111-672), CD172a-PE-Vio770 (130-099-793), CD146-PE-Vio770 (130-099-957), CD344-PE-Vio770 (130-106-572), and CD31-APC (130-110-808) (Miltenyi Biotec). The cells were washed and suspended in PBS, and 7AAD was used to discriminate between living and dead cells. The different samples were then analyzed using a FacsCanto II instrument (Becton Dickinson Biosciences, Le Pont de Claix, France).

Immunophenotypic analyses with flow cytometry were performed according to manufacturer’s recommendations. Briefly, 1 × 10⁶ cells per sample (Passage 3–4) were centrifuged at 300× g and resuspended in 100 µl of buffer. Afterwards, 10 µl of the respective antibodies, CD44-VioBlue, CD73-APC, CD90-PE, CD105-FITC, CD146-PE-Vio770 and CD271-APC-Vio770 (Miltenyi Biotec, Bergisch Gladbach, Germany) and a cocktail of CD14/CD20/CD34/CD45-PerCP (Miltenyi Biotec, Bergisch Gladbach, Germany), was added to cell suspension and incubated for 10 min in the dark in the refrigerator. Then, cells were washed with 2 mL of buffer and centrifuged. Supernatant was aspirated, and the final sediment was resuspended in buffer for flow cytometry analysis. Similarly, respective iso-types controls were used to assess background fluorescence and non-specific binding of anti-bodies to cells. All data were acquired using a MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) and further analyzed by MACS Quantify software (Miltenyi Biotec, Bergisch Gladbach, Germany).