Flow cytometric assay for the detection of GPIIb/IIIa or P-selectin in activated platelets using PAC1 and S12 antibodies. Two FITC-labeled activation-dependent monoclonal antibodies, PAC1 (A and B) or S12 (C and D), were incubated with unactivated platelets (A and C) or to platelets activated by 10 μmol/L thrombin receptor peptide (B and C), and flow cytometry was performed as described in the Materials and Methods. The Y axis displays the number of platelets at any specific fluorescence intensity noted on the X axis as a log scale. The C gate was set at the edge of the background of platelet fluorescence intensity without adding any antibody. The maximal binding of PAC1 or S12 to platelets was defined as the mean fluorescence intensity of platelets activated by 10 μmol/L thrombin receptor peptide and incubated with FITC-PAC1 (80 μg/mL) or FITC-S12 (16 μg/mL). The mean fluorescence intensity obtained in the absence of antibody was similar to that shown in (A) and (C) and was subtracted from that obtained in (B) and (D) to obtain the net mean fluorescence intensity due to PAC1 binding to GPIIb/IIIa or S12 binding to P-selectin on activated platelets.