Gating strategy used to select cell subpopulations for signal-transduction pathway analysis. (A) The CD45-APC-A750/side scatter (SS) pattern was used to isolate lymphocytes from nonlymphocytes, define a generous “blast” region, and exclude nucleated erythrocytes from the analysis. (B) Blasts were further enriched for CD34 and CD117 as shown using CD64-PE and SS characteristics to exclude potential contaminating monocytes and maturing granulocytic cells. (C) CD34 and CD117 profiles from this enriched region were used for defining the CD34-ECD⁺CD117-PC5.5⁺ and CD34-ECD⁻CD117-PC5.5⁺ populations as shown. (D) Mature monocyte and granulocyte enriched regions were defined based on combining the nonlymphocyte gate and CD64-PE and SS light characteristics as shown. (E) The defined granulocyte-enriched region was then further subdivided into immature granulocytes, intermediate granulocytes, and mature granulocytes based on CD13-PC7 and CD16-A700 characteristics as shown. This scheme defines 6 regions (CD34⁺/CD117⁺; CD34⁻/CD117⁺, immature, intermediate and mature granulocytes, and monocytes) from which signaling data were derived. Acquired on Gallios flow cytometer and analyzed with FCS Express Version 3.