Apc labelled annexin 5

MPs were labelled for flow cytometric analysis as previously described.^{30 (link)} Briefly, for the determination of the cellular source of MPs, 25 µL of freshly thawed FFP were mixed with 2.5 µL of fluorescein isothiocyanate (FITC)-labelled anti-CD41 (platelet marker) or phycoerythrin (PE)-labelled anti-CD235a (RBC marker) (BD Biosciences, San Jose, CA). Phosphatidylserine (PS) expression on MPs was determined by labelling 12.5 µL of FFP with 5 µL of allophycocyanin (APC) labelled-annexin-V (BD Biosciences) or FITC labelled-lactadherin (Haematologic Technologies, Essex Junction, Vermont). Samples were diluted with 0.2 µm-filtered phosphate buffered saline (PBS), pH 7.2 or annexin-binding buffer to a final reaction volume of 100 µL in an absolute count tube (TruCount tubes, BD BioSciences). After incubation in the dark at RT for 30 minutes, 300 µL of PBS or annexin-binding buffer was added to each sample. Concentration-matched isotype antibodies were used as controls. Analyses were performed on a FACSCantoII (BD Biosciences) with instrument settings and gating optimized for detection of MPs less than 1 µm diameter as previously described.^{30 (link)} Absolute count of MPs was calculated according to the manufacturer’s instructions (BD Biosciences).