Flow cytometry

To confirm the purity of isolated platelets, the surface expression of the platelet marker CD61 was determined using a specific antibody. Briefly, 1×10⁷ washed platelets were incubated with anti-CD61 monoclonal antibody (GTX61848, Genetex, Irvine, CA, USA) in 0.01% NaN₃ and 1% BSA containing Tyrode’s buffer at 4°C for 1 h, followed by Alexa 594-conjugated goat anti-rabbit IgG secondary antibody (1:500; Thermo Fisher Scientific) incubation for another 30 min.

P-selectin (CD62P) surface expression was determined using FITC-conjugated anti-human CD62P (BD Bioscience, San Diego, CA). Platelets (1×10⁷) were incubated with FITC-conjugated anti-human CD62P (1:10) or FITC-conjugated isotype-matched antibodies (BD Bioscience). PS exposure was detected by staining with a fluorescent conjugate of Annexin V, a protein that has a high affinity for PS, using an Alexa Fluor 594 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

To quantify the binding of NS1 on platelet surfaces, a quantity of 1×10⁷ washed platelets was preincubated with or without an anti-TLR4 antibody (5 μg/ml, Genetex) in 0.1% NaN₃ and 1 μM PGE1 containing Tyrode’s buffer at 4°C for 1 h. NS1 (5 μg/ml) and FITC-conjugated anti-NS1 monoclonal antibodies (33D2-FITC) were added and then incubated at 4°C for another 3 h. The percent fluorescence signal on the platelet surface was analyzed by FACSCalibur flow cytometry. Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).