Ng2 pe

Flow cytometry analysis for hematopoietic and non-hematopoietic cell surface markers was performed on freshly isolated mononuclear cells and on adherent cell populations at p0 and p1. Cells were incubated with single or combinations of the following mouse monoclonal anti-human antibodies in PBS containing 1% fetal calf serum for 30 min at 4 °C: CD45-Alexa 488 (1:20, Beckman Coulter, Nyon, Switzerland), CD45-APC (1:5), CD73-PE-Cy7 (1:40), CD105-PE (1:40), CD90-BV421 (1:13), CD146-PeCy7 (1:20), HLA-DR-PE (1:5), CD19-PE (1:5), CD11b-PE (1:5, all BD Bioscience), CD44-APC (1:10), CD34-PE (1:10, both Miltenyi Biotec, Bergisch-Gladbach, Germany), NG2-PE (1:10), PDGF-rβ-Alexa 700 (1:20, both R&D Systems). Unstained isotype (IgG1-PeCy7 (1:20, BD Bioscience)) and single antibody controls were included. Flow cytometry was performed using a BD Aria III, and a minimum of 25,000 events were acquired for each sample, and data were analyzed using BD FACS Diva 6.1.3. Appropriate compensation settings and gating were applied in order to account for cellular debris, cell doublets, spectral overlap, and autofluorescence.

Cells were labeled for 40 minutes at 4°C with the following antibodies: CD146-APC (1 : 100, R&D Systems, Minneapolis, MN, USA); NG2-PE (1 : 100, R&D Systems); PDGFRβ-PE (1 : 100, R&D Systems); CD31-FITC (1 : 100, BioLegend, San Diego, CA, USA); CD45-APC (1 : 100, BioLegend); CD105-FITC (1 : 100, BioLegend); CD90-PE (1 : 100, BD Biosciences, San Jose, CA, USA); and CD73-PE (1 : 100, BioLegend). Analyses were performed using Guava EasyCyte 5HT™ Flow Cytometer and GuavaSoft 2.1 software (Millipore, Billerica, MA, USA).

For staining, cells were washed with FACS buffer. Antibodies were diluted in 50 μL FACS buffer and added to the cells for 30 minutes at 4°C. After incubation, cells were washed twice with FACS buffer to remove unbound Ab. Cells were then resuspended in FACS buffer and analysed using a FACS Canto II (BD Biosciences). All analysis was performed using FlowJo. The monoclonal antibodies used were: gp38‐PE (e‐Biosciences), Syndecan‐2‐APC and NG2‐PE (R&D), CD105‐PE, MHCII‐FITC, CD29‐PeCy5, CD86‐AF488 (Biolegend), PDGFRα‐PE, CD14‐APC, CD34‐PE, CD15‐FITC, MHCI‐PeCy7, Cd73‐PE, CD90‐PE, CD45‐FITC, CD11b‐PeCy7, CD80‐PeCy7 and CD117‐PE (BD Biosciences).