Influence of citrate concentration on the activation of blood cells in an in vitro dialysis setup
(a) Flow cytometric characterization and quantification of EVs was performed after calibration with fluorescent beads with diameters of 0.1, 0.3, 0.5, and 0.9 μm and the EV gate was set above the 0.9 μm bead cloud and the threshold was set below the 0.3 μm bead cloud to exclude background noise as described in the Methods section. EVs were identified by staining with lactadherin due to their exposure of phosphatidylserine. CD41, CD235a, CD45, and CD14 were used to trace platelet, red blood cell, or monocyte origin of EVs, as shown in representative lactadherin vs. cell surface marker dot plots; (b) increase in EV counts over time. The increase after 120 min was significant for both citrate groups as compared to baseline levels. The difference in EV counts between the two groups was significant at 120 min; (c) cellular origin of EVs in samples at the indicated time points, demonstrating that the large majority of EVs released during recirculation are platelet-derived (lactadherin+CD41+). EVs of undefined origin carry none of the cellular markers (CD41, CD235a, CD45, CD14). Statistically significant differences (p < 0.05) are marked by asterisks.