Infinicyt version 1

EMVs were immunophenotypically defined as CD45−/CD42b−/CD31 + (31). Blood samples were pre-diluted 1:100 in buffered saline solution (PBS) and 50 μL of the diluted solution were used for cell-labeling reaction. Anti-human monoclonal antibodies with suitable volumes were added to the diluted sample: 5 μL of FITC CD45 (BD, clone HI30), 2 μL of CD31 Alexa Fluor 647 (BD, clone WM59) and 5 μL of CD42b PE (BD, clone HIP1). The samples containing monoclonal antibodies were incubated for 30 min protected from light at room temperature, and 2 mL of PBS was added; they were then centrifuged for 5 min at 540 g. The supernatant was discarded and the pellet resuspended with 500 μL PBS and 50 μL counting beads (BD Biosciences, San Jose, California) were added. A total of one million events were acquired using a flow cytometer (FACSCanto II) (BD Biosciences, San Jose, California) and analyzed using by Infinicyt version 1.7 (Cytognos, Salamanca, Spain). A blinded investigator processed all samples. The amount of EMVs was determined from the number and volume of counting beads. Microvesicle sizes were compared to platelet sizes using average fluorescence intensity of forward scatter, showing a mean diameter ≤ 1.0 μm. Figure S5 (supplementary material) describes the gating strategy for CD45−/CD42b−/CD31 + EMVs. EMV was assessed 10 min before and 10 and 70 min post-intervention.

PCs were immunophenotypically defined^{20 (link)} as CD45 + dim/CD34 + and EPCs were defined as CD45 + dim/CD34+/CD309+. Anti-human monoclonal antibodies were added to a 100-μL blood sample at suitable volumes: 3 μL of CD45 PerCP (BD, clone 2D1); 5µL of CD34 PE (BD, clone 8G12); and 5µL of CD309 Alexa Fluor 647 (BD, clone 89,106). Blood samples were incubated protected from light at room temperature for 30 min. Erythrocytes were lysed by addition of 2 mL of 10% BD Pharm Lyse lysing solution (BD Biosciences, San Jose, California) and incubated for 10 min. At least 2 million total events were acquired using a flow cytometer (FACSCanto II) (BD Biosciences, San Jose, California) and analyzed using Infinicyt version 1.7 (Cytognos, Salamanca, Spain). A blinded investigator processed all samples. PCs were calculated as a ratio over CD45 + mononuclear cell counts^{16 (link)} by a gating strategy according to the International Society of Hemotherapy and Graf Engineering (ISHGE) protocol⁵⁹ . Figure S4 (supplementary material) describes the gating strategy used for identifying CD45 + dim/CD34+/CD309 + cells. PC and EPC were assessed 10 min before and 10 and 70 min post-intervention.