Xl system 2 software

After 12-h of fasting, 3 mL of heparinized peripheral blood were obtained and CD34⁺ cells were measured. The precise number of circulating CD34⁺ cells was quantified as we described previously7 (link). We evaluated circulating CD34⁺ cells with a Stem-Kit™ (BeckmanCoulter, Marseille, France) according to the manufacturers’ protocols. These protocols are based on International Society of Hematotherapy and Graft Engineering (ISHAGE) Guidelines8 (link), and are frequently used for quantification of CD34⁺ cells mobilized into the peripheral blood. To increase the reproducibility of CD34⁺ cell counts, the protocol of the Stem-Kit was modified as follows: the blood sample volume, antibodies and lysing solution were doubled. After adding 30 μL of internal control (Stem count: BeckmanCoulter), samples were centrifuged for 5 min at 450 g, and 3,860 μL of supernatant was removed carefully with a pipet. Samples were analyzed by Coulter CYTOMICS™ FC500 and XL-system II software (BeckmanCoulter) for 6 min each.