Attune software

For each blood draw, triplicate samples of 100 µL of heparinized whole blood was stained using the following four-color immunostaining panel: CD31-FITC, CD34-PerCP, CD45-PO, and CD133-PE. For eight of the study participants, a fifth color was added: CD90-v421. Staining was performed as recommended by Thermo Fisher Scientific (Waltham, MA, USA) for whole blood staining followed by a “no-wash” procedure involving Cal-Lyse^® fixation of white blood cells and lysing of red blood cells. In brief, samples were stained in the dark at room temperature for 15 minutes followed by the addition of 100 µL of Cal-Lyse^® Lysing solution and fixation for 10 minutes at room temperature. Red blood cells were then lysed by the addition of 1 mL of deionized water and further 10 minutes incubation in the dark at room temperature. Samples were stored at 4°C in the dark and acquired by flow cytometry within 24 hours using an acoustic-focusing Attune™ flow cytometer (Thermo Fisher Scientific). Files of 300,000–600,000 events were collected for each triplicate sample. Data on stem cell numbers were analyzed by the Attune software (Thermo Fisher Scientific) that provides results as cell per microliter of sample and compensated for the dilution factor that was part of the immunostaining protocol, so the results were converted to stem cell numbers per microliter of whole blood.