Cd45 fitc cd34 pe

To ensure consistent enrichment of CD34⁺/CD117⁺ cell populations to high purity (ie, >90%), CD34⁺/CD117⁺ cells were enriched by using a combination of magnetic cell separation (MACS) and FACS for all samples. Mononuclear cells (MNCs) were extracted by density gradient centrifugation (500g; 20 minutes; Lymphoprep; Pharmacia, Freiburg, Germany) from 40 to 50 mL of heparinized PB (or 2 mL BM), by using 15 mL Biocoll Separating Solution (Biochrom AG, Berlin, Germany). After centrifugation, the interphase with the MNCs was carefully removed, washed in 10 mL phosphate-buffered saline, and centrifuged for another 5 minutes (500g). The cell pellet was recovered and washed in 10 mL phosphate-buffered saline twice. CD34⁺/CD117⁺ cells were isolated from MNCs using MACS by positive selection with the CD34⁺ or CD117⁺ Microbead Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany), according to the manufacturer’s recommendations. For FACS-sorting of CD34⁺/CD117⁺ cells, the CD34- or CD117-enriched fractions were incubated (15 minutes) with the monoclonal antibodies CD45 FITC/CD34 PE (341071; BD Biosciences, San Jose, CA). Sorting of the CD34⁺/CD117⁺ cells was then conducted on a BD FACS Aria II cell sorter (BD Biosciences), aiming for 5 000 to 10 000 CD34⁺/CD117⁺ cells and a purity of >90%.