Figure 1

Schematic of the cold trypsin phosphorylation-specific flow cytometry approach. Cells grown on cell culture dishes at 37°C are treated with activators for the required time and then immediately placed on ice to quench further cell signaling. Ice cold trypsin is added to the cell culture plates and kept on ice during the trypsinization process in order to minimize intracellular signaling. The cells are then harvested, fixed and permeablized prior to staining with optimized phospho-specific primary antibodies and fluorophore-conjugated secondary reagents. Stained cells are analyzed by flow cytometry to quantify single cell fluorescence values corresponding to phosphorylated levels of intracellular signaling proteins.