Mouse platelet aggregation test with embryonic whole blood. Platelets were labeled with CD9-APC or CD9-PE and mixed in 1:1 ratio before stimulation. Aggregation was measured by flow cytometry in time after stimulation with PMA, collagen, Aggretin A, and botrocetin. Average and standard error of the mean are depicted, n > 3. (A) An example of the gating strategy used after flow cytometry analysis. CD9-PE or CD9-APC positive cells (gates APC or PE) were plotted against the forward scatter to select platelets (forward scatter low, named Q1 or Q4 to follow the nomenclature used in the formulas given in “Materials and methods”) or primitive RBC that are also CD9-positive (forward scatter high, pRBC). Note the increase in events in Q2 after stimulation. Within the double-negative gate Q3, Ter119 marked dRBC and the rest were components of blood and plasma that are negative for all markers used (plasma). All these populations are plotted against the forward and side scatter and indicated by colored arrowheads. d, definitive; p, primitive; PLT, platelets; RBC, red blood cells. (B) Time course of double-colored events upon stimulation with PMA, collagen, Aggretin A, and botrocetin. 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).