Peripheral blood samples were collected into heparinized tubes; PBMNCs were then
isolated using Lymphoprep™ tubes (AXIS-SHIELD PoC AS, Oslo, Norway).12 (link) Red cell lysis was performed using BD PharmLyse™ (BD Pharmingen, San
Diego, CA, USA). IgG was blocked for 10 min at room temperature; subsequently,
immunophenotyping of PBMNCs was performed by staining for 15 min on ice in the
dark with fluorochrome-labeled monoclonal antibodies, including anti-human
CD133-FITC (Miltenyi Biotech, Miltenyi Biotec, Bergisch Gladbach, Germany),
VEGFR2-PE (R&D Systems, Minneapolis, MN, USA), CD34-PerCP (BD Pharmingen),
CD90-FITC (BD Pharmingen), PDGFRα-PE (BD Pharmingen), and NGFR-APC (Miltenyi Biotech).13 (link) Isotype-matched control antibodies were used as negative controls to
adjust for fluorochrome overlap.
The fluorescence intensity of cells labeled with fluorochromes was examined using
an Accuri C6 flow cytometer (BD Pharmingen). The data were analyzed using FlowJo
software v10 (BD Pharmingen).
isolated using Lymphoprep™ tubes (AXIS-SHIELD PoC AS, Oslo, Norway).12 (link) Red cell lysis was performed using BD PharmLyse™ (BD Pharmingen, San
Diego, CA, USA). IgG was blocked for 10 min at room temperature; subsequently,
immunophenotyping of PBMNCs was performed by staining for 15 min on ice in the
dark with fluorochrome-labeled monoclonal antibodies, including anti-human
CD133-FITC (Miltenyi Biotech, Miltenyi Biotec, Bergisch Gladbach, Germany),
VEGFR2-PE (R&D Systems, Minneapolis, MN, USA), CD34-PerCP (BD Pharmingen),
CD90-FITC (BD Pharmingen), PDGFRα-PE (BD Pharmingen), and NGFR-APC (Miltenyi Biotech).13 (link) Isotype-matched control antibodies were used as negative controls to
adjust for fluorochrome overlap.
The fluorescence intensity of cells labeled with fluorochromes was examined using
an Accuri C6 flow cytometer (BD Pharmingen). The data were analyzed using FlowJo
software v10 (BD Pharmingen).
