Flow cytometric analysis of PAR-1 density on platelets.
PRP (diluted 1:8, 20 μL) was incubated with 10 μg/mL (final concentration) WEDE15 anti–PAR-1 IgG1 mouse mAb or an irrelevant mouse IgG1 mAb (isotypic control) according to the manufacturer's instructions. After 15 minutes of incubation in the dark, an FITC-labeled antimouse antibody was added (20 μL). After a further 15 minutes in the dark, the mixture was diluted with 2 mL of the kit diluent. In parallel, 40 μL of calibration beads were stained with 20 μL FITC-labeled antimouse antibody, then diluted. Sample and calibration tubes were stored for 30 minutes at room temperature in the dark before flow cytometric analysis of 5000 events (FACScan flow cytometer; Becton Dickinson). (A) Histogram representing calibration bead fluorescence. The mean fluorescence intensity (M) of each peak was reported on a calibration curve. One representative example is shown (log-log scale, r2 = 0.998). The coefficient of variation of 100 curve slopes was 1.2%. (B) Typical histogram of PAR-1 labeling with WEDE15 mAb (solid line) and an isotypic control (dotted line). The number of specific PAR-1 sites was calculated after subtracting the negative isotypic control value with a calculation template (Biocytex).
