Quantification of Circulating Progenitor Cells

Circulating CD34+/CD133+/CD45^low cells, including EPCs were measured by flow cytometry using minor modifications of a previously described method [16 (link)–18 (link)]. In brief, EDTA-treated peripheral blood was incubated with the test or control reagent. The reagent mixture consisted of nucleic acid dye (SY-III-8; Molecular Probe, Eugene, OR, USA), peridinine chlorophil protein (PerCP)-conjugated anti-CD45 (Becton Dickinson, San Jose, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (Becton Dickinson) and phycoerythrin (PE)-conjugated anti-CD133 (Miltennyi Biotec, Bergisch Gladbach, Germany). Isotype controls were used as the negative controls based on the species and immunoglobulin (Ig) G control antibodies (IgG1 isotype control; Becton Dickinson). Flow cytometric analysis was then performed using a FACS Calibur laser flow cytometer (Becton Dickinson) according to the manufacturer’s instructions. Each measurement consisted of 10⁶ events of all white blood cells, which exceeded the threshold set for SY-III-8 fluorescence (nucleated cells). The absolute number of CD34+/CD133+/CD45^low cells per mL was calculated based on the cells-to-the whole-blood cell count.