Anti pac 1 fitc

The polyelectrolyte multilayer films were prepared from low molecular weight cationic chitosan (Chi) and anionic chondroitin sulfate (CS) purchased from Sigma-Aldrich (Saint Louis, MO, USA). Cross-linking chemicals i.e., 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) and N-hydrosulfosuccinimide (NHS) were purchased from Sigma-Aldrich. The human blood was purchased from a donation center. A plasma protein adsorption assay was performed using Qubit^® Protein Assay kit from ThermoFischer Scientific (Waltham, MA, USA). An impact R test kit was purchased from DiaMed Ltd. (Sofia, Bulgaria). Blood coagulation system activation was determined based on a confocal laser scanning microscopy and a flow cytometry analysis after staining with anti-CD62P FITC, anti-CD45 PE, anti-PAC-1 FITC, anti-CD61-PerCP, anti-CD14-PerCP, and anti-CD61-FITC antibodies as well as isotype controls (anti-IgG1-PE, anti-IgM-FITC, and anti-IgG1-FITC) supplied by BD Bioscience (San Jose, CA, USA). A ZymuphenMP-activity ELISA kit from Hyphen Biomed Eragny (Neuville-sur-Oise, France), was applied in a microparticles concentration analysis.

The expression of platelet surface markers CD62p and PAC-1 were examined using flow cytometry (BD, FACSCanto plus, USA). The protocol has been previously described by Morel et al. [21 (link)]. Briefly, a part of whole blood samples were activated by ADP (20 µM) at 37 °C for 10 min. Resting and activated samples were fixed in 1% paraformaldehyde (PFA) at 37 °C for 1 h and stained with the corresponding antibodies: anti-CD61/PerCP, anti-CD62p/PE and anti-PAC-1/FITC (BD,San Diego, CA, USA) in dark at room temperature for 30 min. Then, samples were diluted in PBS until further use. Flow cytometry analysis was performed on 10,000 platelets (CD61/PerCP-positive). The results were presented as percentages of CD62p- and PAC-1-positive platelets in the samples.