Pe conjugated anti human cd31

To quantify the levels of EMPs and PMPs within samples, PPP samples were mixed with 10 μm flow counting beads (Beckman Coulter, UK) in the presence of calcium-rich buffer (eBioscience, UK). Analysis of the MP populations was carried out using a cocktail of phycoerythrin (PE) -conjugated anti-human CD31 (BD Bioscience, UK), allophycocyanin (APC) -conjugated anti-human CD42b (BD Bioscience, UK) and efluor450 annexin-V marker (eBioscience, UK) as previously described34 (link). MPs were analysed using a Cyan flow cytometer (BD Biosciences) according to their size and fluorescence using a logFS-logSS plot. EMPs were characterised and enumerated as Annexin V+/CD31+/CD42b- events, and PMPs as Annexin V+/CD31+/CD42b+ events (Supplementary Fig. S1).

CD31 expressing cells were isolated on day 8 of differentiation via magnetically activated cell sorting (MACS; Miltenyi Biotec Bergisch Gladbach, Germany) following the manufacturer’s protocol. After washing with 1× phosphate-buffered saline (PBS, ThermoFisher Scientific), cells were harvested with TrypLE Express and resuspended in MACS buffer (0.5 EDTA and 0.5% BSA in PBS). Cells were then incubated with 10 μl of PE-conjugated anti-human CD31 (BD Biosciences, San Jose, CA) for 10 min at 4 °C. After incubation, the unbound primary antibody was removed by washing with MACS buffer twice. Next, 20 μl of anti-PE microbeads (Miltenyi Biotec Bergisch Gladbach, Germany) were added to 80 μl of cells suspended in MACS buffer and incubated for an additional 15 min at 4 °C. Cells were washed with MACS buffer and separated using the MS MACS separation column (Miltenyi Biotec). Following separation, CD31 and VECAD enrichment were confirmed using flow cytometry as previously^{71 (link)} and detailed below in a separate section. Finally, CD31+ cells were seeded on type I collagen-coated plates and maintained in EC differentiation media. For all experiments, media was switched to ECGM without growth factor supplementation for 24 h unless otherwise noted. All experiments used iECs between passages 1 and 3.