A rapid screening method for monitoring signaling changes in the monocyte cell line U937 (AACR 2009)

Here we describe a precise screening assay that quantifies changes in phosphorylation of proteins in samples from as few as 100 cells, which is simple, rapid, and relatively low in cost. A nano-immunoassay system (Cell Biosciences) was used to measure changes in expression and activation of relevant signaling proteins, including MEK, ERK and STATs in U937 monocyte cells before and after cytokine treatment. A single pan-specific antibody was used to distinguish between the phosphorylated and non-phosphorylated protein isoforms, as the nano-immunoassay (NIA) method separates different phosphorylated forms of a protein based on their isoelectric point. In parallel, phospho-protein FACS analysis, which is the current state of the art for measuring multiple signaling pathways, was performed to compare changes in expression and phosphorylation of the signaling proteins. Phospho-protein FACS analysis is expensive and requires considerable technical expertise, which limits its application for large numbers of samples. This novel nano-immunoassay screening method is currently being employed at the Stanford Human Immune Monitoring Core (HIMC) and is being used for high-throughput screening of compounds that influence monocyte activation, monocyte/macrophage differentiation and analysis of various disease states in small primary tissue samples. Practical examples will be given.