Early EPCs were characterized from the uptake of 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine-labeled acetylated low-density Lipoprotein (DiI-Ac-LDL) and lectin binding. Staining was performed by incubating EPCs with 10 mg/mL of DiI-Ac-LDL (Invitrogen, Life Technologies, Ltd., Paisley, UK) for 2 hours at 37°C. Then, cells were fixed in 4% paraformaldehyde for 30 min and counterstained with 1 mg/mL FITC-labelled lectin from Ulex europaeus (UEA-1-FITC) (Sigma-Aldrich, Ltd.) for 2 hours at 37°C in the dark. Images of the stained cells were viewed with a fluorescence microscope and double-positive DiI-Ac-LDL/lectin cells were preliminarily identified as early EPCs [23 (link)].
To evaluate the immunophenotype of EPCs, adherent cells were detached with trypsin-EDTA and 5 × 105 cell/tube were incubated with anti-human CD34-PE (BD Biosciences), CD133-PE (Miltenyi Biotec), VEGFR-2-Alexa Fluor 647 (BioLegend), CD31-FITC (BD Biosciences), CD45-FITC (BD Biosciences), and CD42-PE (BD Biosciences) antibodies for 30 min in the dark at 4°C. Isotype control antibodies were used to set baseline fluorescence levels. The labeled cells were analyzed on a FACSCalibur Instrument (BD Biosciences), acquiring 2 × 104 events for each analysis. The flow cytometric analysis was repeated six times.
To evaluate the immunophenotype of EPCs, adherent cells were detached with trypsin-EDTA and 5 × 105 cell/tube were incubated with anti-human CD34-PE (BD Biosciences), CD133-PE (Miltenyi Biotec), VEGFR-2-Alexa Fluor 647 (BioLegend), CD31-FITC (BD Biosciences), CD45-FITC (BD Biosciences), and CD42-PE (BD Biosciences) antibodies for 30 min in the dark at 4°C. Isotype control antibodies were used to set baseline fluorescence levels. The labeled cells were analyzed on a FACSCalibur Instrument (BD Biosciences), acquiring 2 × 104 events for each analysis. The flow cytometric analysis was repeated six times.