Human platelet aggregation test with washed platelets. (A) Dot plots depicting platelets labeled with CFSE or PKH26 mixed in 1:1 ratio as measured by flow cytometry before addition of PMA (t = 0 min, left) or upon PMA stimulation (t = 10 min, right). Platelets stained with PKH26 are enclosed in quadrant Q1. Platelets stained with CFSE are enclosed in quadrant Q4. Double-colored events in quadrant Q2 represent aggregates before and after stimulation by PMA. All populations are plotted against the forward and side scatter and indicated by colored arrowheads. PLT, platelet. (B) Images of single-stained platelets from quadrants Q1 and Q4 or aggregates from quadrant Q2 obtained with ImageStreamX are depicted next to the respective dot plots. BF, bright field. (C) Platelets were labeled with CFSE or PKH26 and mixed in 1:1 ratio before stimulation. Aggregation was measured by flow cytometry in time after stimulation with PMA, collagen, Aggretin A, or ristocetin alone or in the presence of either the integrin β3 inhibitor tirofiban or blocking anti-integrin β1 antibody LIA 1/2.1. Average and standard error of the mean are depicted, n > 3. Nonstimulated labeled platelets are shown as baseline for spontaneous aggregation. Colored asterisks at each data point indicate statistical significance with the unstimulated control (P < .05).
