Cd42b pe cy5

Whole blood was diluted 1:5 with HEPES saline buffer and incubated with a cocktail of the following fluorescent conjugated antibodies at saturating concentrations: PAC‐1 fluorescein (FITC), CD62P phycoerythrin (PE), CD42b PE‐Cy5, or IgG1Κ PE isotype control (all BD Pharmingen) for exactly 15 min. Six reaction tubes were used that were identical in function and agonist concentrations as those used for MPAs. However, ADP at the concentration used (1.5 μmol/L) caused maximal PAC‐1 binding in all participants, so was not included in statistical analysis. Following incubation, samples were fixed with 800 μL of stabilizing fixative (BD Biosciences) and were then stored at 4°C until analysis by flow cytometry (BD FACSCanto II) within 24 h. Samples were run at a low flow rate until 10,000 platelet positive events were counted. To account for spectral overlap between the three fluorophores, single‐stained compensation beads were used (BD Biosciences). For both MPAs and platelet surface receptor binding, samples were incubated at room temperature with the exception of tubes containing AA and collagen, which were incubated at 37°C using a dry block heater (Ratek DBH20D, Victoria, Australia).

CD42b receptor transfer to neutrophils and monocytes cell surface was investigated by flow cytometry using a Beckman Coulter Cytoflex (Beckman). Citrated blood from five healthy donors was incubated with 0.5 μg/ml E. coli O111:B4 LPS (Sigma-Aldrich®) for 15 min at 37 °C. 100 μl of blood was used and red blood cells were lysed with a BD Phosflow™ Lyse/Fix 5X. After lysis, samples were washed once with 0.5% BSA in PBS and incubated with CD66b-FITC (Clone: G10F5, BD Bioscience), CD42b-PE-Cy™5 (Clone: HIP1, BD Bioscience) and CD14-BV421 (Clone: HCD14, BioLegend®) antibodies were added (1:50) to the cell suspension and incubated for 60 min at 37 °C in the dark. CD42b is platelet-specific surface protein as claimed by the manufacturer. To exclude that other cells can up-regulate CD42b upon stimulation, isolated neutrophils were stimulated with LPS. For one donor, the manual gating analysis using FMO, and gating controls was carried out. Samples were washed once with 0.5% BSA in PBS and the cell pellet was resuspended in 300 μl of washing buffer. The percentage of CD42b on the cell surface was calibrated with control cells that were not treated with LPS. The results are presented as mean values ± s.e.m.