Pac 1 fitc

After treatment (in vitro: metformin: 1 mM, 6 h, 37 °C; aspirin: 0.1 mM, 6 h, 37 °C; mtDNA: 40 ng/μL, 6 h, 37 °C. In vivo: metformin: 200 mg/kg twice a day for 7 days; aspirin: 15 mg/kg twice a day for 7 days; mtDNA: 25 μg/kg twice a day for 7 days), platelets were incubated with PE-CD62P (Sigma, USA) or PAC-1-FITC (Sigma, USA) for 30 min in the dark, and reaction was stopped by adding ice-cold PBS. The P-selectin and integrin-αIIbβ3 expression on the platelets was measured at 585 nm (FL2) and 530 nm (FL1) by using flow cytometer (Becton - Dickinson, San Jose, CA, USA). In selected experiments platelets were pre-incubated for 0.5 h at 37 °C with neutralizing antibodies against DC-SIGN (120507; R&D Systems, USA, 25 μg/mL) or DNase I (Genentech, USA, 20 μg/mL)33 (link)45 (link).

Ten microlitres of platelet-specific antibodies (PAC-1-FITC, CD62p-PE and CD36-PE [Becton Dickson (BD), USA]) were used in the detection of the platelet membrane glycoproteins (GPIIb/IIIa, P- Selectin and GPIV respectively). PAC1- FITC and CD62p-PE were double stained while CD36- PE was single stained. 5μL Arginylglycylaspartic acid (RGD) [Sigma—Aldrich, USA] solution was added in the staining mixture of PAC-1-FITC and CD62p-PE to demonstrate specific PAC-1 binding.
Equal volumes (2.5 μL) of inactivated and activated blood samples were stained with the antibodies and incubated for 15 min at room temperature in the dark. 500 μL of the cold 1% paraformaldehyde solution (BD, USA) was used to fix the stained cells for flow cytometry analysis.