Development Of A Flow Cytometry Phospho-STAT5 Assay In Nonhuman Primate T Cells

By Robyn Pryor, Megan Templeton, Cory Chew, Brandon Purdy, Skylar Brown, Ryan Karkas, MaKinna Van Horn, Elizabeth Sulpy, Catherine Schilffarth, and Martin Poirier

Phosphorylated STAT5 (pSTAT5) plays a crucial role in T cell activation and proliferation, making its quantification a valuable tool for assessing T cell function, particularly in toxicology studies. IL-2 activates T cells through its receptor, leading to the phosphorylation of STAT5, which is essential for immune responses. Flow cytometry has emerged as a powerful method for detecting pSTAT5, offering advantages over traditional techniques like western blotting by enabling precise, high-throughput analysis of multiple cell populations. While flow cytometry pSTAT5 assays have been developed for use in cell lines, no such assay has been established in nonhuman primates (NHPs) for toxicological purposes.

This poster outlines the development of a flow-cytometry-based pSTAT5 assay in Macaca fascicularis. Using peripheral blood from six naïve macaques, we stimulated cells with recombinant IL-2 and quantified pSTAT5 levels across various T cell populations. Key aspects of method development include assay feasibility, reproducibility, and comparisons between PB and PBMCs, along with fluorescence controls for accuracy.