Effect of TNF-α and APC on MP-associated EPCR release. (A) HUVECs (light gray bars) and monocytes (dark gray bars) were incubated with TNF-α (10 ng/mL) or APC (100 nM) or both for 2 hours and analyzed by flow cytometric analysis with the percentage of EPCR surface expression expressed from the mean value of 3 experiments ± SD with the asterisk indicating P < .005. Isolated MPs from the conditioned media were quantified by ELISA through (B) APC detection via chromogenic S2366 (nM) and (C) EPCR content (ng/mL). Results are representative of 3 experiments. Values are mean ± SD; *P < .005. (D) Flow cytometric analysis and quantification of MPs released from cultured monocytes: (i) determination of forward (FSC) and side-scatter (SSC) using 1-μm beads to establish the MP gate (R1); (ii) R2 gate includes the 7-μm beads used for enumeration of MPs; (iii) determination of the limit of negative fluorescence in the presence of EDTA as a negative control for annexin V and the IgG-PE isotypic control; and (iv) MPs dually labeled with annexin V–FITC and anti–EPCR-PE.