Analyzing Platelet Surface Phenotypes

To identify the cellular phenotype of platelets, expression of surface molecules was examined by flow cytometry. For gating purposes, the platelet population was verified using anti-CD41a-VioBlue (Clone REA386; Miltenyi Biotec, Auburn, CA, USA) monoclonal antibody (mAb) staining. Antibodies used to probe surface molecules included anti-P-selectin-FITC (Clone CLBThromb/6; Beckman Coulter, Westbrook, ME, USA), anti-TLR2-Alexa Fluor 488 (Clone T2.5; Abcam), anti-TLR4-APC (Clone HTA125; Abcam) and anti-TLR9-FITC (Clone 5G5; Abcam) mAbs. Isotype-matched control IgGs for each mAb were used to set negative populations. Platelets were analyzed on a MACS Quant Analyzer (Miltenyi Biotec) and data were analyzed using FlowJo v.7.6.4 software (Tree Star, Ashland, OR, USA). Details of the gating strategies of platelets are shown in Fig. S1. As shown in Fig. S1, TLR9 is found in intracellular domains, though it has been reported that TLR9 could migrate to the outer membrane after activation^{34 (link)}. TLR9 expression was measured after permeabilization of the cells using cytofix/cytoperm permeabilization kit (BD Biosciences, San Jose, CA, USA).