Quantification of intracellular proteins and monitoring therapy using flow cytometry

Here we review phospho-specific, quantitative flow cytometry approach as a rapid and reliable tool for measuring intracellular signaling proteins with potential applications in monitoring efficacy of targeted therapy. The single cell, multiparameter nature of flow cytometry allows simultaneous investigation of specific cell type and the corresponding intracellular markers. Peripheral blood can be directly stained with surface markers to delineate cell populations of interest, followed by fixation, permeabilization, and immunostaining with specific antibodies to the cellular targets. By using calibrated standardized phycoerythrin (PE)-conjugated beads for signal quantification, an informative Index value can be generated for each sample by multiplication of percentages of positive cells with fluorescence intensity per cell. This technique can yield both qualitative and quantitative information on effects of cellular markers upon targeted therapy, thereby providing another layer of advantages over the conventional flow cytometry analysis. Advances in this technology: high-throughput capability and automation, making it a valuable platform in modern drug discovery.