Quantifying Endothelial Microparticles by Flow Cytometry

Amounts of EMPs were measured using the flow cytometry method [4 (link),18 (link)]. In the assay, EA.hy926 cells were pre-treated with EA and Eth extracts (50–200 µg/ml) for 1 h and incubated with TNF-α (10 ng/ml) for 24 h. After incubation, the culture medium was collected and centrifuged at 5000×g for 15 min. The supernatant was collected and centrifuged at 20000×g for 40 min. The supernatant was discarded, and the EMPs pellet was reconstituted in Annexin V binding buffer and stained with FITC-Annexin V for 15 min in the dark at room temperature. Subsequently, CountBright™ counting beads were added in each sample as an internal control and 5000 counting bead events were analysed using FACSCalibur Flow Cytometer (BD) with BD CellQuest Pro Software (BD Biosciences, San Jose, CA, U.S.A.). For EMPs gating, calibrated latex beads (1.1 µm diameter) were used to set the population of particle size < 1 µm. The gated populations which stained with FITC-Annexin V were used to set the subpopulation of positive Annexin V EMPs. The numbers of positive Annexin V EMPs were normalised with the counting beads and were presented as absolute numbers of EMPs as well as the fold-change of EMPs relative to the control.